AU2019351809C1 - Biodegradable lipids for the delivery of active agents - Google Patents
Biodegradable lipids for the delivery of active agentsInfo
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- AU2019351809C1 AU2019351809C1 AU2019351809A AU2019351809A AU2019351809C1 AU 2019351809 C1 AU2019351809 C1 AU 2019351809C1 AU 2019351809 A AU2019351809 A AU 2019351809A AU 2019351809 A AU2019351809 A AU 2019351809A AU 2019351809 C1 AU2019351809 C1 AU 2019351809C1
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- lipid
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- peg
- alkyl
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- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1617—Organic compounds, e.g. phospholipids, fats
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- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
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- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
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- C07C217/08—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to an acyclic carbon atom
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Description
WO wo 2020/072324 PCT/US2019/053617 PCT/US2019/053617
This application claims the benefit of U.S. Provisional Application No. 62/739,548, filed
on October 1, 2018, the entire contents of which are hereby incorporated by reference.
The present invention relates to biodegradable lipids and to their use for the delivery of
active agents such as nucleic acids.
Therapeutic nucleic acids include, e.g., small interfering RNA (siRNA), micro RNA
(miRNA), antisense oligonucleotides, ribozymes, plasmids, immune stimulating nucleic acids,
antisense, antagomir, antimir, microRNA mimic, supermir, U1 adaptor, and aptamer. In the case
of siRNA or miRNA, these nucleic acids can down-regulate intracellular levels of specific proteins
through a process termed RNA interference (RNAi). The therapeutic applications of RNAi are
extremely broad, since siRNA and miRNA constructs can be synthesized with any nucleotide
sequence directed against a target protein. To date, siRNA constructs have shown the ability to
specifically down-regulate target proteins in both in vitro and in vivo models. In addition, siRNA
constructs are currently being evaluated in clinical studies.
However, two problems currently faced by siRNA or miRNA constructs are, first, their
susceptibility to nuclease digestion in plasma and, second, their limited ability to gain access to
the intracellular compartment where they can bind the protein RISC when administered
systemically as the free siRNA or miRNA. Lipid nanoparticles formed from cationic lipids with
other lipid components, such as cholesterol and PEG lipids, and oligonucleotides (such as siRNA
and and miRNA) miRNA)have been have used been to facilitate used the cellular to facilitate uptake of the cellular the oligonucleotides. uptake of the oligonucleotides.
-1-
There remains remainsa aneed need for for improved cationic lipids and lipid nanoparticles for the 01 Apr 2025 2019351809 01 Apr 2025
There improved cationic lipids and lipid nanoparticles for the
delivery of oligonucleotides. delivery of oligonucleotides.Preferably, Preferably, these these lipid lipid nanoparticles nanoparticles wouldwould provideprovide high high drug:lipid ratios, protect the nucleic acid from degradation and clearance in serum, be suitable drug:lipid ratios, protect the nucleic acid from degradation and clearance in serum, be suitable
for for systemic delivery, systemic delivery, andand provide provide intracellular intracellular delivery delivery of the of the nucleic nucleic acid. Inacid. In addition, addition, these these lipid-nucleic acidparticles lipid-nucleic acid particlesshould should be be well-tolerated well-tolerated and provide and provide an adequate an adequate therapeutic therapeutic index, index, such that patient such that patient treatment treatment atat an aneffective effective dose doseofofthe thenucleic nucleicacid acidis isnotnotassociated associated with with
significant toxicityand/or and/or risk to to the patient. 2019351809
significant toxicity risk the patient.
The present The present invention inventionrelates relates to to improved lipid particles improved lipid particles as as well well as as improved cationic improved cationic
lipids. lipids.
Accordingtotoaa first According first aspect, aspect,the thepresent invention present provides invention providesa a compound of formula compound of (A): formula (A):
R¹ z¹ R R * X 1 N a b Q M R'
R² Z²
Formula (A) Formula (A)
or a salt or a salt thereof, wherein thereof, wherein
R’ is absent, R' is absent,hydrogen, hydrogen, or alkyl; or alkyl;
with respect to R and R2, 1 R², with respect to R¹ and
(i) R1 and (i) R¹ andR²R2are areeach, each,independently, independently, optionally optionally substituted substituted alkyl,alkenyl, alkyl, alkenyl, alkynyl, cycloalkylalkyl, alkynyl, cycloalkylalkyl, heterocycle, heterocycle, or R10; or R¹;
(ii) R¹1 and (ii) R R2,together andR², together with with thethe nitrogen nitrogen atom atom to which to which they arethey are attached, attached, form form an optionallysubstituted an optionally substituted heterocylic heterocylic ring;ring; or or
(iii) (iii)one one of R¹1 and of R 2 an optionally substituted alkyl, alkenyl, alkynyl, andR²Ris is an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkyl, cycloalkylalkyl, or orheterocycle, heterocycle,and andthe theother otherforms formsa a4-10 4-10membered heterocyclic membered heterocyclic
-2- ring or or heteroaryl heteroaryl with with (a) (a) the the adjacent adjacent nitrogen nitrogen atom and(b) (b) the the (R)a (R)a group groupadjacent adjacenttotothe the 01 Apr 2025 2019351809 01 Apr 2025 ring atom and nitrogen atom; nitrogen atom; each occurrenceofof RRis, each occurrence is, independently, –(CR3R4)-; independently, -(CR³R)-; each each occurrence R3and occurrenceofof R³ 4 andRRare, are,independently independentlyH, H, halogen, halogen, OH,OH, alkyl, alkyl, alkoxy, alkoxy, -NH2, -NH,
R10,alkylamino, R¹, alkylamino,orordialkylamino; dialkylamino; 2019351809
10 independently selected from PEG and polymers based on each occurrenceofofR¹Ris each occurrence is independently selected from PEG and polymers based on poly(oxazoline), poly(ethylene poly(oxazoline), oxide), poly(vinyl poly(ethylene oxide), poly(vinyl alcohol), alcohol), poly(glycerol), poly(glycerol), poly(N- poly(N- vinylpyrrolidone), vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] poly[N-(2-hydroxypropyl)methacrylamide] and and poly(amino poly(amino acid)s, acid)s, wherein wherein
(i) (i) the the PEG or polymer PEG or polymerisislinear linearororbranched, branched,(ii) (ii) the the PEG PEGor or polymer polymer is polymerized is polymerized by n by n
subunits, subunits, (iii) (iii)n is a number-averaged n is a number-averageddegree degree of ofpolymerization polymerization between 10and between 10 and200 200units, units, and and 10 (iv) (iv) the thecompound hasatat most compound has mosttwo twoR¹Rgroups; groups;
the dashed line to Q is absent or a bond; the dashed line to Q is absent or a bond;
whenthe when thedashed dashedline linetotoQ Qisisabsent absentthen thenQ Q is is absentororisis-0-, absent -O-,-NH-, -NH-, -N(R5-S-, -N(R)-, )-, -S-, - - C(O)-, C(O)-, -C(O)O-, -C(O)O-, -OC(O)-, -C(O)N(R4)-, -OC(O)-, -C(O)N(R)-, -N(R5)C(O)-, -N(R)C(O)-, -S-S-, -S-S-, -OC(O)O-, -OC(0)0-, -O-N=C(R-5)-, - -O-N=C(R)-, 5 C(R C(R)=N-O-, -OC(O)N(R5-N(R²)C(O)N(R³), )=N-O-,-OC(O)N(R)-, )-, -N(R5)C(O)N(R 5 )-, -N(R5)C(O)O-, -N(R°)C(O)O-, -C(O)S-, -C(O)S-, -C(S)O--C(S)O- or - or - 5 C(R )=N-O-C(O)-;or C(R³)=N-O-C(O)-; or
whenthe when thedashed dashedline linetotoQ Qisisa abond bondthen then(i)(i)b bisis 00 and and(ii) (ii) Q andthe Q and thetertiary tertiary carbon carbon
adjacent adjacent totoitit(C*) (C*)form form a substituted a substituted or unsubstituted, or unsubstituted, mono- mono- or or bi-cyclic bi-cyclic heterocyclic heterocyclic group group having from having from55toto 10 10 ring ring atoms; atoms;
5 each each occurrence is, independently, occurrenceofof RRis, independently,HHororalkyl; alkyl;
X is alkylene or alkenylene; X is alkylene or alkenylene;
M1is M¹ is aa biodegradable group; biodegradable group;
aa is is 1,1, 2,2,3,3,4,4,5 5or or 6; 6;
b is 0, 1, 2, or 3; b is 0, 1, 2, or 3;
Z1 is Z¹ is aaCC-C 6-Cbranched 14 branched alkyl alkyl group; group; andand
- 2A 2A--
Z2 is is CC-C 4-Calkenyl, 20 alkenyl, wherein thethe alkenyl group maymay optionally be substituted with oneone 01 Apr 2025 2019351809 01 Apr 2025
Z² wherein alkenyl group optionally be substituted with
or or two fluorine atoms two fluorine at the atoms at the alpha alpha position positiontotoa a double doublebond bond which which is isbetween between the the double bond double bond
and theterminus and the terminus Z².Z2. of of
Accordingtotoaa second According secondaspect, aspect, the the present present invention invention provides provides aa compound compound selected selected
from: from: 2019351809
0
0
- 2B 2B -
2019351809 01 Apr 2025
N 2019351809
or a salt or a salt thereof. thereof.
According to a third aspect, the present invention provides a lipid particle comprising According to a third aspect, the present invention provides a lipid particle comprising
aa neutral lipid, aa lipid neutral lipid, lipid capable capableofofreducing reducing aggregation, aggregation, and a and a cationic cationic lipid lipid of of a compound a compound of of the first or second aspects. the first or second aspects.
According toaa fourth According to fourth aspect, aspect, the the present present invention invention provides provides aa pharmaceutical pharmaceutical
composition comprising composition comprising a lipid a lipid particle particle of theof the third third aspectaspect and a pharmaceutically and a pharmaceutically acceptable acceptable
carrier. carrier.
Accordingtotoaa fifth According fifth aspect, aspect,the thepresent presentinvention inventionprovides providesa amethod method of of modulating the modulating the
expression expression ofof a a target target gene gene in aincell, a cell, thethe method method comprising comprising providing providing to athe to the cell cell a lipid lipid
particle of the third aspect. particle of the third aspect.
According to a sixth aspect, the present invention provides a method of treating a According to a sixth aspect, the present invention provides a method of treating a
disease ordisorder disease or disordercharacterized characterized by overexpression by the the overexpression of a polypeptide of a polypeptide in athe in a subject, subject, the methodcomprising method comprising providing providing to to thesubject the subjecta apharmaceutical pharmaceutical composition composition of of thethe fourth fourth
aspect, wherein aspect, wherein thethe active active agent agent is a is a nucleic nucleic acid acid selected selected from from the theconsisting group group consisting of an of an siRNA, siRNA, a amicroRNA, microRNA,and and an antisense an antisense oligonucleotide, oligonucleotide, and and wherein wherein the the siRNA, siRNA, microRNA, microRNA,
- 2C 2C-- or antisenseoligonucleotide oligonucleotide includes a polynucleotide that specifically binds to binds to a 01 Apr 2025 2019351809 01 Apr 2025 or antisense includes a polynucleotide that specifically a polynucleotide that polynucleotide that encodes the polypeptide, encodes the polypeptide, or or aa complement thereof. complement thereof.
Accordingtotoaa seventh According seventhaspect, aspect, the the present present invention invention provides a method provides a oftreating method of treating aa disease ordisorder disease or disordercharacterized characterized by underexpression by underexpression of a polypeptide of a polypeptide in athe in a subject, subject, the methodcomprising method comprising providing providing to to thesubject the subjecta apharmaceutical pharmaceutical composition composition of of thethe fourth fourth
aspect, wherein aspect, wherein thethe active active agent agent is a is a plasmid plasmid that encodes that encodes the polypeptide the polypeptide or a functional or a functional 2019351809
variant or fragment thereof. variant or fragment thereof.
Accordingtotoan According aneighth eighthaspect, aspect, the the present present invention invention provides provides a a method of inducing method of inducinganan immune response immune response in in a a subject,the subject, the method methodcomprising comprising providing providing to to thethe subjecta a subject
pharmaceuticalcomposition pharmaceutical compositionofof thefourth the fourthaspect, aspect, wherein whereinthe theactive active agent agent is is an an
immunostimulatory oligonucleotide. immunostimulatory oligonucleotide.
According to a ninth aspect, the present invention provides the use of a lipid particle According to a ninth aspect, the present invention provides the use of a lipid particle
as defined in the third aspect in the manufacture of a medicament for treating a disease or as defined in the third aspect in the manufacture of a medicament for treating a disease or
disorder characterized disorder characterized by by the the overexpression overexpression of a polypeptide, of a polypeptide, wherein wherein the active the active agent is a agent is a
nucleic acid nucleic acid selected selected from from the the group group consisting consisting of of an an siRNA, siRNA, aa microRNA, microRNA, andand an an antisense antisense
oligonucleotide, and oligonucleotide, whereinthe and wherein the siRNA, siRNA,microRNA, microRNA, or antisense or antisense oligonucleotide oligonucleotide includes includes a a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a polynucleotide that specifically binds to a polynucleotide that encodes the polypeptide, or a
complement complement thereof. thereof.
According to a tenth aspect, the present invention provides the use of a lipid particle According to a tenth aspect, the present invention provides the use of a lipid particle
as definedininthe as defined thethird thirdaspect aspect in in thethe manufacture manufacture of a medicament of a medicament for atreating for treating disease a ordisease or
disorder characterized disorder characterized by by underexpression underexpression of a polypeptide, of a polypeptide, wherein wherein the active the active agent is a agent is a
plasmidthat plasmid that encodes the polypeptide encodes the polypeptideoror aa functional functional variant variant or or fragment fragment thereof thereof
According to an eleventh aspect, the present invention provides the use of a lipid According to an eleventh aspect, the present invention provides the use of a lipid
particle as defined in the third aspect in the manufacture of a medicament for inducing an particle as defined in the third aspect in the manufacture of a medicament for inducing an
immune response,wherein immune response, wherein thethe activeagent active agentisisananimmunostimulatory immunostimulatory oligonucleotide. oligonucleotide.
One embodiment is a lipid particle comprising a biodegradable cationic lipid, a neutral One embodiment is a lipid particle comprising a biodegradable cationic lipid, a neutral
lipid, aasterol, lipid, sterol,and anda a lipid capable lipid ofofreducing capable reducingaggregation aggregation(e.g., (e.g.,PEG-modified lipid), where PEG-modified lipid), where
the molar ratio of the biodegradable cationic lipid to the sterol ranges from about 1.6:1 to about the molar ratio of the biodegradable cationic lipid to the sterol ranges from about 1.6:1 to about
- 2D 2D--
2.0:1 and/or the molar ratio of the biodegradable cationic lipid to the neutral lipid ranges from 01 Apr 2025 2019351809 01 Apr 2025
2.0:1 and/or the molar ratio of the biodegradable cationic lipid to the neutral lipid ranges from
about 5.5:1 about 5.5:1 to to about 5.9:1. The about 5.9:1. Theinventors inventorshave havesurprisingly surprisinglyfound found thatlipid that lipidparticles particles having having certain higher certain contents of higher contents of biodegradable biodegradablecationic cationiclipid lipidrelative relative to to the the amount amountsterol steroland/or and/or neutral lipid exhibit enhanced efficacy for the delivery of an active agent (e.g., siRNA). In one neutral lipid exhibit enhanced efficacy for the delivery of an active agent (e.g., siRNA). In one
embodiment, thebiodegradable embodiment, the biodegradable cationiclipid cationic lipidcomprises comprisesa alipid lipid moiety, moiety,where wherethe thelipid lipid moiety moiety has one has one or or more morebiodegradable biodegradablegroups groups (such (such as as an an estergroup ester group (-C(O)O- (-C(O)O- or –OC(O)-). or -0C(0)-). In In one one preferred embodiment, embodiment,the the lipid capable of reducing aggregation is 1,2-dimyristoyl-sn- 2019351809
preferred lipid capable of reducing aggregation is 1,2-dimyristoyl-sn-
glycerol-methoxy polyethylene glycerol-methoxy polyethylene glycol glycol(PEG-DMG), suchasasPEG-DMG (PEG-DMG), such PEG-DMGwith with an average an average
polyethyleneglycol polyethylene glycol molecular molecularweight weightofof2000. 2000.
In a further embodiment, the molar ratio of the biodegradable cationic lipid to the sterol In a further embodiment, the molar ratio of the biodegradable cationic lipid to the sterol
is is from from about 1.7 to about 1.7 to about about 1.9:1, 1.9:1, such such as as about about 1.9:1. 1.9:1. In In another another embodiment, themolar embodiment, the molarratio ratio of the biodegradable of the biodegradable cationic cationic lipidlipid to neutral to the the neutral lipid lipid rangesranges from5.5:1 from about about 5.5:15.8:1, to about to about 5.8:1, such as about 5.8:1. such as about 5.8:1.
- 2E 2E -
PCT/US2019/053617
In one embodiment, the lipid particle comprises from about 55 to about 60 mol % of the
biodegradable cationic lipid, such as about 58 mol % (based on 100 mol % of the lipid components
in the lipid particle). The lipid particle may comprise from about 28 to about 33 mol % of the
sterol, such as from about 28 to about 32 mol % (based on 100 mol % of the lipid components in
the lipid particle). In one embodiment, the lipid particle comprises from about 3 to about 12 mol
%, such as from about 5 to about 12 mol %, from about 8 to about 12 mol %, or from about 9 to
about 11 mol %, of the neutral lipid (based on 100 mol % of the lipid components in the lipid
particle). In another embodiment, the lipid particle comprises about 10 mol % of the neutral lipid
(based on 100 mol % of the lipid components in the lipid particle). In yet another embodiment,
the lipid particle comprises from about 0.5 to about 10 mol %, such as from about 0.5 to about 5
mol % or from about 1 to about 3 mol %, of the lipid capable of reducing aggregation (e.g., a PEG-
modified lipid) (based on 100 mol % of the lipid components in the lipid particle).
Another embodiment is a lipid particle comprising a biodegradable cationic lipid, a neutral
lipid, a sterol, and a lipid capable of reducing aggregation (e.g., PEG-modified lipid), where the
lipid particle comprises from about 55 to about 60 mol % of the biodegradable cationic lipid and
from about 33 to about 28 mol % of the sterol (based on 100 mol % of the lipid components in the
lipid particle). In one embodiment, the lipid particle comprises about 58 mol % of the
biodegradable cationic lipid (based on 100 mol % of the lipid components in the lipid particle). In
another embodiment, the lipid particle comprises from about 3 to about 12% of the neutral lipid,
and from about 0.5 to about 10 mol % of the lipid capable of reducing aggregation (based on 100
mol % of the lipid components in the lipid particle). In yet another embodiment, the lipid particle
comprises about 10 mol % of the neutral lipid (based on 100 mol % of the lipid components in the
lipid particle). In yet another embodiment, the lipid particle comprises about 2 mol % of the lipid
capable of reducing aggregation (based on 100 mol % of the lipid components in the lipid particle).
In one embodiment, the lipid particle comprises from about 55 to about 60 mol % of the cationic
lipid, from about 3 to about 12% of the neutral lipid, from about 28 to about 33 mol % of the sterol,
and from about 0.5 to about 10 mol % of the lipid capable of reducing aggregation (based on 100
mol % of the lipid components in the lipid particle). In yet another embodiment, the lipid particle
comprises about 58% of the cationic lipid, about 10% of the neutral lipid, about 30% of the sterol,
and about 2% of the lipid capable of reducing aggregation (based on 100 mol % of the lipid
PCT/US2019/053617
components in the lipid particle). In yet another embodiment, the lipid particle comprises about
55% of the cationic lipid, about 10% of the neutral lipid, about 33% of the sterol, and about 2% of
the lipid capable of reducing aggregation (based on 100 mol % of the lipid components in the lipid
particle). In one preferred embodiment, the lipid capable of reducing aggregation is 1,2-
dimyristoyl-sn-glycerol-methoxy polyethylene glycol (PEG-DMG), such as PEG-DMG with an
average polyethylene glycol molecular weight of 2000.
One embodiment is a cationic lipid having the formula (A):
R¹ Z1 Z¹ R' NR1 R N R Rb b ** a X M1 Q M R'
R² R2 Z2 Z²
Formula (A)
or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein
R' is absent, hydrogen, or alkyl (e.g., C1-C4 alkyl); C-C alkyl);
with withrespect to R Superscript(1) respect to R¹ and R², and R2,
(i) R R¹¹ and and R² R2 are are each, each, independently, independently, optionally optionally substituted substituted alkyl, alkyl, alkenyl, alkenyl, alkynyl, alkynyl,
cycloalkylalkyl, cycloalkylalkyl, heterocycle, or R10; heterocycle, or R¹;
(ii) (ii) RR¹ Superscript(1) and R2, with and R², together together the with the nitrogen nitrogen atom toatom to which which theythey are are attached,form attached, form an an
optionally substituted heterocylic ring; or
(iii) one of R1 R¹ and R2 R² is optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkylalkyl, or heterocycle, and the other forms a 4-10 membered heterocyclic ring or
heteroaryl (e.g., a 6-member ring) with (a) the adjacent nitrogen atom and (b) the (R)a group
adjacent to the nitrogen atom;
each occurrence of R is, independently, -(CR3R)-- -(CR³R)-; wo 2020/072324 WO PCT/US2019/053617 PCT/US2019/053617 each each occurrence occurrenceof of R³ R³ and and R4 are, independently R are, H, halogen, independently OH, alkyl, H, halogen, OH,alkoxy, alkyl,-NH2, R 10,-NH, R¹, alkoxy, alkylamino, or dialkylamino (in one preferred embodiment, each occurrence of R3 R³ and R4 are, R are, independently H or C1-C4 alkyl); C-C alkyl); each occurrence of R10 is independently R¹ is independently selected selected from from PEG PEG and and polymers polymers based based on on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized by n subunits,
(iii) n is a number-averaged degree of polymerization between 10 and 200 units, and (iv) wherein
the compound of formula has at most two R10 groups (preferably R¹ groups (preferably at at most most one one R¹ R10 group); group);
the dashed line to Q is absent or a bond;
when the dashed line to Q is absent then Q is absent or is -O-, -0-, -NH-, -N(R5)-, -S-,-C(O)-, -N(R)-, -S-, -C(O)-,
-C(O)O-, -OC(O)-, -C(0)0-, -OC(O)-,-C(O)N(R4)-, -C(O)N(R)-,-N(R5)(())-, -S-S-, -N(R)C(O)-, -OC(O)O-, -S-S-, -O-N=C(R5)-, -0C(0)0-, -C(R5)=N-O-, -O-N=C(R)-, -C(R)=N-O-, - -
OC(O)N(R)-, -N(R°)C(O)N(R²)-, -N(R°)C(O)O-, -C(O)S-, OC(O)N(R5)-,-N(R5)C(O)N(R5)-,-N(R5)C(O)O-,-C(O)S-, -C(S)O- -C(S)O- or or -C(R³)=N-O-C(O)-; -C(R5)=N-O-C(0)-; or or
when the dashed line to Q is a bond then (i) b is 0 and (ii) Q and the tertiary carbon adjacent
to it (C*) form a substituted or unsubstituted, mono- or bi-cyclic heterocyclic group having from
5 to 10 ring atoms (e.g., the heteroatoms in the heterocyclic group are selected from O and S,
preferably O);
each each occurrence occurrenceof of R5 R is, independently, is, H or H independently, alkyl (e.g. C1-C4 or alkyl (e.g.alkyl); C-C alkyl);
X X is is alkylene alkyleneoror alkenylene (e.g., alkenylene C4 toCC20 (e.g., to alkylene or C4ortoC C20 C alkylene to alkenylene); C alkenylene);
M¹ is a biodegradable group (e.g., -OC(O)-, -C(O)O-, M1 -C(0)0-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-
, , -S-S-, -S-S-, -C(R5)=N-, -C(R)=N-, -N=C(R5)-, -N=C(R)-, -C(R5)=N-O-, -C(R)=N-O-,-O-N=C(R5)-, -O-N=C(R)-,-C(O)(NR5)-, -C(O)(NR)-,-N(R3)(()), -N(R)C(O)-, - C(S)(NR)-, -N(R)C(O)-, C(S)(NR5)-, -N(R²)C(O)N(R²)-, -N(R5)(()), -OC(O)O-, -0C(0)0-, -OSi(R)O-, -OSi(R5)20-, -C(O)(CR³R)C(O)O-, - -C(0)(CR'R')C(O)0-, O-R¹¹ NVV
OC(O)(CR²R)C(O)-, oror OC(O)(CR?R*)C(O)-, O (wherein (whereinR R¹¹ 11 is isa aC2-C8 C-C alkyl alkylororalkenyl)); alkenyl));
y a is 1, 2, 3, 4, 5 or 6;
WO wo 2020/072324 PCT/US2019/053617
b is 0, 1, 2, or 3;
Z Z¹¹ is is a a C6-C14 branched alkyl C-C branched alkyl group; group; and and
Z² is a C4-C20 alkenyl, C-C alkenyl, wherein wherein thethe alkenyl alkenyl group group maymay optionally optionally be be substituted substituted with with oneone
or two fluorine atoms at the alpha position to a double bond which is between the double bond and
F F S my the terminus of Z² (e.g., ).
The R'R¹R²N-(R)a-Q-(R)- group can be any of the head groups described herein, The group can be any of the head groups described herein, including those shown in Table 1 below, and salts thereof. In one preferred embodiment,
R'R'R?N-(R)A-Q-(R)6- R'R¹R²N-(R)a-Q-(R)- is (CH3)2N-(CH2)2-, is (CH)N-(CH)-, (CH3)2N-(CH2)3-C(O)O-, (CH)N-(CH)-C(O)O-, (CH3)2N-(CH2)2-NH- (CH)N-(CH)-NH- C(O)O-, C(O)O-,(CH3)2N-(CH2)2-OC(O)-NH-, (CH)N-(CH)-OC(O)-NH-, or or (CH3)2N-(CH2)3-C(CH3)=N-O- (CH)N-(CH)-C(CH)=N-O-. In a In preferred a preferred embodiment, R'R¹R²N-(R)a-Q-(R)- is (CH)N-(CH)-. embodiment, is (CH3)2N-(CH2)2-. In In one oneembodiment, embodiment,R Superscript(1) R¹ and R² are andboth R2 are both (e.g., alkyl alkyl (e.g., methyl, methyl, ethyl ethyl or or a combination thereof). a combination thereof).
In In one oneembodiment, R Superscript(1) embodiment, R¹ and R² andare R2 are bothmethyl. both methyl. InIn another embodiment, another one of R Superscript(1) embodiment, one of R¹ and and R² R2 is ismethyl methyl
and the other of R R¹¹ and and R² R2 is is ethyl. ethyl.
In a further embodiment, a is 2. In another embodiment, b is 0. In another embodiment Q
is absent. In yet another embodiment, a is 2, b is 0 and Q is absent. In yet another embodiment, a
is 4, b is 0 and Q is -O-. -0-.
In another embodiment, X is -(CH2)n- wherein -(CH)- wherein n n isis 4 4 toto 20, 20, e.g., e.g., 4 4 toto 18, 18, 4 4 toto 16, 16, oror 4 4 toto 12. 12.
In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one embodiment, X is -(CH2)7-9-. -(CH)-9-. InIn one one exemplary exemplary
embodiment, embodiment,X Xisis -(CH2)7-. -(CH)-.InInone oneexemplary embodiment, exemplary X is X embodiment, -(CH2)8-. In one is -(CH)-. In exemplary one exemplary
embodiment, embodiment,X Xisis -(CH2)9-. -(CH)-.
In further embodiments, M M¹¹is is-OC(O)- -OC(O)-or or-C(0)0-. -C(O)O-.For Forexample, example,in inone oneembodiment, embodiment,M¹ M ¹
is -C(O)O-. -C(0)0-. In another embodiment, M M¹¹ is is -OC(O)-. -OC(O)-.
In In another anotherembodiment, Z Superscript(1) embodiment, Z¹ is a C-C is branched a C6-C10 branched alkyl alkyl group, group, e.g., e.g., -CH(CH2CH3)(CH2CH2CH2CH3), -CH2CH('Pr)(CH2CH2'Pr) or -CH(CHCH)(CHCHCHCH), -CHCH('Pr)(CHCH'Pr) or -CH2CH(n-Bu)2. -CHCH(n-Bu). - - 6 -
WO wo 2020/072324 PCT/US2019/053617 PCT/US2019/053617
In another embodiment, Z² is C19 alkenyl containing one or two double bonds. For example, In another embodiment, Z² is C alkenyl containing one or two double bonds. For example,
isB-(CH2)9CH=CHCH2CH=CH(CH2)4CH3 Z2 Z is -(CH)CH=CHCHCH=CH(CH)4CH. Yet another embodiment is a cationic lipid selected from: Yet another embodiment is a cationic lipid selected from:
0
a
N i
- - 7 -
In N.
- -8 -
WO wo 2020/072324 PCT/US2019/053617
and salts thereof (e.g., pharmaceutically acceptable salts thereof).
Yet embodiment is a compound having the formula (A-I):
O u V S
W it t q
Formula (A-I)
or a salt thereof (e.g., pharmaceutically acceptable salt thereof);
wherein S, s, t, u, V and q are each, independently, 0, 1, 2, 3, 4, 5, 6 or 7; and
WO wo 2020/072324 PCT/US2019/053617
W is a head group (e.g., a protonatable amine group having a pKa of between about 4 and
about 11, such as between about 4 and about 7, between about 5 and about 7, or between about
5.5 and about 6.8).
Suitable head groups include any of those described herein (see, e.g., Table 1A).
In In certain certainembodiments, embodiments,the the headhead groupgroup is (CH3)2N-(CH2)2-, is (CH)N-(CH)-,(CH3)2N-(CH2)3-C(O)O-, (CH)N-(CH)-C(O)O-,
(CH3)2N-(CH2)2-NH-C(O)O-, (CH3)2N-(CH2)2-OC(O)-NH-, (CH)N-(CH)-NH-C(O)O-, (CH)N-(CH)-OC(O)-NH-,oror(CH3)2N-(CH2)3-C(CH3)=N-O-. (CH)N-(CH)-C(CH)=N-O-. In In one one embodiment, embodiment,thethe head group head is (CH3)2N-(CH2)2-- group is (CH)N-(CH)-.
In one embodiment, variable S is 3 to 5, such as 4.
In one embodiment, variable t is 4 to 6, such as 5.
In one embodiment, variable q is 2 to 4, such as 3.
In one embodiment, variable u is 0 to 2, such as 1.
In one embodiment, variable V is 0 to 2, such as 1.
Yet another embodiment is a compound having the formula (A-II):
O 100 R100 R
R200 N R² b
Formula (A-II)
or a salt thereof (e.g., pharmaceutically acceptable salt thereof) wherein
b is 0, 1, 2 or 3;
S is 0, 1, 2, 3, 4 or 5;
R200 R² isis C12-C22 alkyl, C12-C22 C-C alkyl, alkyenyl, or C12-C22 C-C alkyenyl, or C-Calkynyl; and alkynyl; and
- 10
WO wo 2020/072324 PCT/US2019/053617 PCT/US2019/053617
R R 100 100isis C5-C15 C-C alkyl, alkyl,C5-C15 C-C alkyenyl, alkyenyl,or C5-C15 or C-Calkynyl. alkynyl.
In one embodiment, b is 1.
In one embodiment, S is 2, 3, or 4, such as 3.
In In one oneembodiment, R 100 R¹ embodiment, is ais C8-C12 a C-Calkyl. alkyl.
In In one oneembodiment, R200 is embodiment, R²C12-C14 is C-C alkyl, C12-C14 alkyl, C-Calkyenyl, or C12-C14 alkyenyl, or C-C alkynyl. alkynyl.
In In another anotherembodiment, R200 isR² embodiment, C18-C20 alkyl, is C-C C18-C20 alkyl, alkyenyl, C-C or C18-C20 alkyenyl, alkynyl. or C-C alkynyl.
These cationic lipids described herein can be incorporated into lipid particles. One
embodiment is a lipid particle comprising a cationic lipid, such as those described above (including
those of formula (A), (A-I) and (A-II)). Each of the cationic lipids described herein includes one
or more biodegradable groups. The biodegradable groups are located in a lipidic moiety (e.g., a
hydrophobic chain) of the cationic lipid. These cationic lipids may be incorporated into a lipid
particle for delivering an active agent, such as a nucleic acid (e.g., an siRNA). The incorporation
of the biodegradable group(s) into the lipid results in faster metabolism and removal of the lipid
from the body following delivery of the active agent to a target area. As a result, these lipids have
lower toxicity than similar lipids without the biodegradable groups.
The lipid particles described herein may further comprise an active agent. Non-limiting
examples of active agents include nucleic acids, such as plasmids, immunostimulatory
oligonucleotides, siRNAs, antisense oligonucleotides, microRNAs, antagomirs, aptamers, and
ribozymes. In one preferred embodiment, the nucleic acid is an siRNA.
The lipid particles described herein may be incorporated into a pharmaceutical
composition. One embodiment is a pharmaceutical composition comprising a lipid particle
described herein and a pharmaceutically acceptable carrier. The lipid particle preferably includes
an active agent, such as a nucleic acid. In one preferred embodiment, the active agent is an siRNA.
Yet another embodiment is a method of modulating the expression of a target gene in a
cell, comprising providing to the cell a lipid particle described herein. In one embodiment, the
active agent is a nucleic acid is an siRNA.
- 11
WO wo 2020/072324 PCT/US2019/053617
Yet another embodiment is a method of treating a disease or disorder characterized by the
overexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical
composition described herein. In one embodiment, the active agent is a nucleic acid selected from
the group consisting of an siRNA, a microRNA, and an antisense oligonucleotide, and wherein the
siRNA, microRNA, or antisense oligonucleotide includes a polynucleotide that specifically binds
to a polynucleotide that encodes the polypeptide, or a complement thereof.
Yet another embodiment is a method of treating a disease or disorder characterized by
underexpression of a polypeptide in a subject, comprising providing to the subject a
pharmaceutical composition described herein. In one preferred embodiment, the active agent is a
plasmid that encodes the polypeptide or a functional variant or fragment thereof.
Yet another embodiment is a method of inducing an immune response in a subject,
comprising providing to the subject a pharmaceutical composition described herein. In one
preferred embodiment, the active agent is an immunostimulatory oligonucleotide.
Figure 1 is a bar graph depicting the relative Factor VII protein levels following
administration of the lipid formulations described in Example 4.
Figure 2 is a bar graph depicting the relative Factor VII protein levels following
administration of the lipid formulations described in Example 4.
Figure 3 is a bar graph depicting the relative Factor VII protein levels following
administration of the lipid formulations described in Example 4.
Figure 4 is a bar graph depicting the relative Factor VII protein levels following
administration of the lipid formulations described in Example 4.
Figure 5 is a graph depicting the relative Factor XII plasma levels following administration
of the lipid formulations described in Example 5.
- 12
PCT/US2019/053617
Figure 6A is a graph depicting the relative Factor XII plasma levels following
administration of the lipid formulations AF-094 (0.03, 0.1, and 0.3 mg/kg) and AF-011 (0.3 mg/kg)
described in Example 6.
Figure 6B is a graph depicting the relative Factor XII plasma levels following
administration of the lipid formulations AF-079 (0.3 mg/kg) and AF-011 (0.3 mg/kg) described in
Example 6.
Figure 7 is a graph depicting the relative Factor XII plasma levels following administration
of the lipid formulations AF-073 (0.03, 0.1, and 0.3 mg/kg) and AF-011 (0.3 mg/kg) described in
Example Example 6. 6.
Figure 8 is a graph depicting the relative Factor XII plasma levels following administration
of the lipid formulations AF-093 (0.03, 0.1, and 0.3 mg/kg) and AF-011 (0.3 mg/kg) described in
Example Example 6. 6.
Figure 9 is a graph depicting the relative Factor XII plasma levels following administration
of the lipid formulations AF-083 (0.1 and 0.3 mg/kg) and AF-011 (0.3 mg/kg) described in
Example 6.
The Cationic The CationicLipid Lipid
The cationic lipid can be any biodegradable cationic lipid known in the art, such as those
described describedininInternational InternationalPublication Nos. Nos. Publication WO 2011/153493, WO 2013/086322, WO 2011/153493, WO WO 2013/086322, WO 2013/086354, and WO 2013/086373, U.S. Patent Nos. 9,012,498, 9,061,063, and 9,463,247, and
U.S. Patent Publication No. 2014/0308304, which are hereby incorporated by reference in their
entirety. These biodegradable cationic lipids include one or more biodegradable groups. This
results in faster metabolism and removal of the cationic lipid from the body following delivery of
the active agent to a target area. As a result, these cationic lipids have substantially lower toxicity
than similar cationic lipids without the biodegradable groups. In one embodiment, one or more
- 13 wo 2020/072324 WO PCT/US2019/053617 biodegradable groups are located in the mid- or distal section of a lipidic moiety (e.g., a hydrophobic chain) of the cationic lipid.
In one embodiment, the biodegradable cationic lipid has the formula (A):
R1 R¹ z¹ R R * X N a Q b M1 M R'
R2 R² Z2 Z²
Formula (A)
or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein
R' is absent, hydrogen, or alkyl (e.g., C1-C4 alkyl); C-C alkyl);
R¹ and R2, with respect to R1 R²,
(i) (i) RR¹ Superscript(1) and R² are and R2 are each, each, independently, independently, optionally optionally substituted substituted alkyl, alkyl, alkenyl, alkynyl, alkenyl, alkynyl,
cycloalkylalkyl, cycloalkylalkyl, heterocycle, or R10: heterocycle, or R¹;
(ii) (ii) RR¹ Superscript(1) and R², with and R², together together the with the nitrogen nitrogen atom toatom to which which theythey are are attached,form attached, form an an
optionally substituted heterocylic ring; or
(iii) (iii) one oneofof R Superscript(1) R¹ and R² is and R2 is optionally optionally substituted substituted alkyl,alkyl, alkenyl, alkenyl, alkynyl,cycloalkyl, alkynyl, cycloalkyl,
cycloalkylalkyl, or heterocycle, and the other forms a 4-10 membered heterocyclic ring or
heteroaryl (e.g., a 6-member ring) with (a) the adjacent nitrogen atom and (b) the (R)a group
adjacent to the nitrogen atom;
-(CR³R)-; each occurrence of R is, independently, -(CR3R)--
each each occurrence occurrenceof of R³ R³ and and R4 are, independently R are, H, halogen, independently OH, alkyl, H, halogen, OH, alkoxy, alkyl, -NH2, R 10,-NH, R¹, alkoxy,
alkylamino, or dialkylamino (in one preferred embodiment, each occurrence of R3 R³ and R4 are, R are,
independently H or C1-C4 alkyl); C-C alkyl);
- 14 wo 2020/072324 WO PCT/US2019/053617 each occurrence of R10 is independently R¹ is independently selected selected from from PEG PEG and and polymers polymers based based on on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] poly[N-(2-hydroxypropyl)methacrylamide) and poly(amino acid)s, wherein (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized by n subunits,
(iii) n is a number-averaged degree of polymerization between 10 and 200 units, and (iv) wherein
the compound of formula has at most two R10 groups (preferably R¹ groups (preferably at at most most one one R¹ R10 group); group);
the dashed line to Q is absent or a bond;
-0-, -NH-, -N(R5)-, when the dashed line to Q is absent then Q is absent or is -O-, -N(R)-, -S-, -S-,-C(O)-, -C(O)-,
-C(O)O-, -OC(O)-, -C(0)0-, -OC(O)-,-C(O)N(R4)-, -C(O)N(R)-,N(R5)(())-, -S-S-, -N(R)C(O)-, -OC(O)O-, -S-S-, -O-N=C(R5)-, -0C(0)0-, -C(R5)=N-O-, -O-N=C(R)-, - -C(R³)=N-O-,
OC(O)N(R5)-, OC(O)N(R)-, -N(R5)C(O)N(R5)-, -N(R°)C(O)N(R²)-,-N(R5)((())-, -C(O)S-, -N(R°)C(O)O-, -C(S)O- -C(O)S-, or -C(R5)=N-O-C(0)-; -C(S)O- or or -C(R³)=N-O-C(O)-; or
when the dashed line to Q is a bond then (i) b is 0 and (ii) Q and the tertiary carbon adjacent
to it (C*) form a substituted or unsubstituted, mono- or bi-cyclic heterocyclic group having from
5 to 10 ring atoms (e.g., the heteroatoms in the heterocyclic group are selected from O and S,
preferably O);
each each occurrence occurrenceof of R5 R is, independently, is, H or H independently, alkyl (e.g. C1-C4 or alkyl (e.g.alkyl); C-C alkyl);
X X is is alkylene alkyleneor or alkenylene (e.g., alkenylene C4 to CC20 (e.g., to alkylene or C4ortoC C20 C alkylene to alkenylene); C alkenylene);
M1 M¹ is a biodegradable group (e.g., -OC(O)-, -C(O)O-, -C(0)0-, -SC(O)-, -C(O)S-, -OC(S)-, -C(S)O-
, -S-S-, -C(R5)=N-, -S-S-, -C(R)=N-, -N=C(R5)-, -N=C(R)-, -C(R5)=N-O-, -C(R)=N-O-,-O-N=C(R5)-, -O-N=C(R)-,-C(O)(NR5)-, -C(O)(NR)-,-N(R5)(()), -N(R)C(O)-, - C(S)(NR)-, -N(R)C(O)-, C(S)(NR5)-, -N(R°)C(O)N(R³)-, -N(R5)(()), -OC(O)O-, -0C(0)0-, -OSi(R)O-, -OSi(R5)20-, -C(O)(CR³R)C(O)O-, - -C(O)(CR^R*)C(0)0-,- O-R¹¹ O-R11 NVV
OC(O)(CR'R*)C(O)-,on OC(O)(CR²R)C(O)-, or O (wherein (whereinR R¹¹ 11 is isa aC2-C8 C-C alkyl alkyl or oralkenyl)); alkenyl));
a is 1, 1, 2,2,3,3,4, 4, 55 or or 6; 6;
O, 1, 2, or 3; b is 0,
C-C branched Z¹ is a C6-C14 alkyl branched group; alkyl andand group;
WO wo 2020/072324 PCT/US2019/053617
Z2 Z² is a C4-C20 alkenyl, C-C alkenyl, wherein wherein thethe alkenyl alkenyl group group maymay optionally optionally be be substituted substituted with with oneone
or two fluorine atoms at the alpha position to a double bond which is between the double bond and
F, F F S my the terminus of Z² (e.g., ).
The R'R¹R²N-(R)a-Q-(R)- group can be any of the head groups described herein, The group can be any of the head groups described herein, including those shown in Table 1 below, and salts thereof. In one preferred embodiment,
R'R¹R²N-(R)a-Q-(R)- is is (CH)N-(CH)-,(CH3)2N-(CH2)3-C(O)O-, (CH3)2N-(CH2)2-, (CH)N-(CH)-C(O)O-, (CH)N-(CH)-NH- (CH3)2N-(CH2)2-NH-
C(O)O-, (CH3)2N-(CH2)2-OC(O)-NH- C(O)O-, (CH)N-(CH)-OC(O)-NH-, oror (CH3)2N-(CH2)3-C(CH3)=N-O- (CH)N-(CH)-C(CH)=N-O-. InInaa preferred preferred embodiment, R'R¹R²N-(R)a-Q-(R)- is (CH)N-(CH)-. embodiment, is (CH3)2N-(CH2)2-- In one embodiment, R1 R¹ and R2 R² are both alkyl (e.g., methyl, ethyl or a combination thereof).
In In one oneembodiment, embodiment,R1 R¹ and and R2 are R² both methyl. are both In another methyl. embodiment, In another one of R Superscript(1) embodiment, one of R¹ andand R²R2isismethyl methyl
and the other of R Superscript(1) and R2 is ethyl. and the other of R¹ and R² is ethyl.
In a further embodiment, a is 2. In another embodiment, b is 0. In another embodiment Q
is absent. In yet another embodiment, a is 2, b is 0 and Q is absent. In yet another embodiment, a
is 4, b is 0 and Q is -O-. -0-.
In another embodiment, X is -(CH2)n -(CH)n- wherein n is 4 to 20, e.g., 4 to 18, 4 to 16, or 4 to
12. In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one embodiment, X is -(CH2)7-9-. -(CH)-9-. InIn one one
exemplary embodiment, X is -(CH2)7-. -(CH)-. InIn one one exemplary exemplary embodiment, embodiment, X X isis -(CH2)- -(CH)-. InIn one one
exemplary exemplaryembodiment, embodiment,X is X -(CH2)9-. is -(CH)-.
In further embodiments, M M¹¹ is is -OC(O)- -OC(O)- or or -C(0)0-. -C(O)O-. For For example, example, in in one one embodiment, embodiment, M¹ M ¹
is -C(O)O-. -C(0)0-. In another embodiment, M M¹¹is is-0C(0)-. -OC(O)-.
In In another anotherembodiment, Z Superscript(1) embodiment, Z¹ is a C-C is branched a C6-C10 branched alkyl alkyl group, group, e.g., e.g., -CH(CH2CH3)(CH2CH2CC3), CH2CH(Pr)(CH2CH2Pr) or or -CH(CHCH)(CHCHCHCH3),-CHCH(Pr)(CHCHPr) -CH2CH(n-Bu)2. -CHCH(n-Bu),
Z² is a C19 In another embodiment, Z2 C alkenyl having alkenyl one having oror one two double two bonds. double For bonds. example, For example,
Z2 can be Z can be -(CH)øCH=CHCHCH=CH(CH)CH. -(CH2)9CH=CHCH2CH=CH(CH2)4CH3
WO wo 2020/072324 PCT/US2019/053617
Yet Yet another another embodiment embodiment is is a a cationic cationic lipid lipid selected selected from: from:
0
0
program
PCT/US2019/053617
and salts thereof (e.g., pharmaceutically acceptable salts thereof).
In certain embodiments, the biodegradable group present in the cationic lipid is selected
from an ester (e.g., -C(O)O- or -OC(O)-), disulfide (-S-S-), oxime (e.g., -C(H)=N-O- or -O- -0-
N=C(H)-), -C(0)-0-,-OC(0)-, -C(R5)=N-, -C(O)-O-,-OC(O),-C(R²)=N- -N=C(R5)-, -N=C(R)-, -C(R5)=N-O-, -C(R)=N-O-, -O-N=C(R5)-, -O-N=C(R)-, -O-C(O)O-, -0-C(0)0-,
-C(O)N(R5),-N(R)C(O)-, -C(O)N(R), -N(R5)((C)-, -C(S)(NR5)-, -C(S)(NR)-, (NR3)(C)S), (NR)C(S)-, -N(R5)C(O)N(R5)-, -N(R°)C(O)N(R³)-, -C(O)S-, -C(O)S-, -SC(O)-, -SC(O)-,
-C(S)O-, -OC(S)-, -C(S)O-, -OC(S)-,-OSi(R5)20-, -OSi(R)O-,-C(0)(CR?R*)C(O)0-, -C(O)(CR³R)C(O)O-,or or -OC(O)(CR³R)C(O)-.
In one embodiment, the aliphatic group in one or both of the hydrophobic tails of the
cationic lipid includes at least one carbon-carbon double bond.
A suitable cholesterol moiety for the cationic lipids (including compounds of formulas (A),
(A-I) and (A-II) has the formula:
H Illiii Illli
Additional embodiments include a cationic lipid having a head group, one or more
hydrophobic tails, and a linker between the head group and the one or more tails. The head group
can include an amine; for example, an amine having a desired pKa. The pKa can be influenced by
WO wo 2020/072324 PCT/US2019/053617 PCT/US2019/053617
the structure of the lipid, particularly the nature of head group; e.g., the presence, absence, and
location of functional groups such as anionic functional groups, hydrogen bond donor functional
groups, hydrogen bond acceptor groups, hydrophobic groups (e.g., aliphatic groups), hydrophilic
groups (e.g., hydroxyl or methoxy), or aryl groups. The head group amine can be a cationic amine;
a primary, secondary, or tertiary amine; the head group can include one amine group (monoamine),
two amine groups (diamine), three amine groups (triamine), or a larger number of amine groups,
as in an oligoamine or polyamine. The head group can include a functional group that is less
strongly basic than an amine, such as, for example, an imidazole, a pyridine, or a guanidinium
group. The head group can be zwitterionic. Other head groups are suitable as well.
The one or more hydrophobic tails can include two hydrophobic chains, which may be the
same or different. The tails can be aliphatic, for example, they can be composed of carbon and
hydrogen, either saturated or unsaturated but without aromatic rings. The tails can be fatty acid
tails. Some such groups include octanyl, nonanyl, decyl, lauryl, myristyl, palmityl, stearyl,
a-linoleyl, -linoleyl, stearidonyl, stearidonyl,linoleyl, y-linolenyl, linoleyl, arachadonyl, -linolenyl, and oleyl. arachadonyl, and Other oleyl.hydrophobic tails are tails are Other hydrophobic
suitable as well.
The linker can include, for example, a glyceride linker, an acyclic glyceride analog linker,
or a cyclic linker (including a spiro linker, a bicyclic linker, and a polycyclic linker). The linker
can include functional groups such as an ether, an ester, a phosphate, a phosphonate, a
phosphorothioate, a sulfonate, a disulfide, an acetal, a ketal, an imine, a hydrazone, or an oxime.
Other linkers and functional groups are suitable as well.
In one embodiment, the cationic lipid is a racemic mixture. In another embodiment, the
cationic lipid is enriched in one diastereomer, e.g. the cationic lipid has at least 95%, at least 90%,
at least 80% or at least 70% diastereomeric excess. In yet another embodiment, the cationic lipid
is enriched in one enantiomer, e.g. the lipid has at least 95%, at least 90%, at least 80% or at least
70% enantiomer excess. In yet another embodiment, the cationic lipid is chirally pure, e.g. is a
single optical isomer. In yet another embodiment, the cationic lipid is enriched for one optical
isomer.
Where a double bond is present (e.g., a carbon-carbon double bond or carbon-nitrogen
double bond), there can be isomerism in the configuration about the double bond (i.e. cis/trans or
- 20
E/Z isomerism). Where the configuration of a double bond is illustrated in a chemical structure, it
is understood that the corresponding isomer can also be present. The amount of isomer present can
vary, depending on the relative stabilities of the isomers and the energy required to convert
between the isomers. Accordingly, some double bonds are, for practical purposes, present in only
a single configuration, whereas others (e.g., where the relative stabilities are similar and the energy
of conversion low) may be present as inseparable equilibrium mixture of configurations.
In some cases, a double-bonded unsaturation can be replaced by a cyclic unsaturation. The
cyclic unsaturation can be a cycloaliphatic unsaturation, e.g., a cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl group. In some cases, the cyclic group can be
a polycyclic group, e.g., a bicyclic group or tricyclic group. A bicyclic group can be bridged, fused,
or have a spiro structure.
In some cases, a double bond moiety can be replaced by a cyclopropyl moiety, e.g.,
can be replaced by / For example, the moiety shown below has two carbon-
carbon double bonds, each of which can independently be replaced by a cyclic moiety, e.g., a
cyclopropyl moiety. Thus, substitutes for: O can include:
O O , and and
-21-
WO wo 2020/072324 PCT/US2019/053617
For further example, substitutes for I
O m include:
For further example, substitutes for my COOMe
include: COOMe
For further example, substitutes for COOEt 3 include: COOEt
The cationic lipid includes one or more biodegradable groups. The biodegradable group(s)
include one or more bonds that may undergo bond breaking reactions in a biological environment,
e.g., in an organism, organ, tissue, cell, or organelle. Functional groups that contain a
biodegradable bond include, for example, esters, dithiols, and oximes. Biodegradation can be a
factor that influences the clearance of the compound from the body when administered to a subject.
Biodegredation can be measured in a cell based assay, where a formulation including a cationic
lipid is exposed to cells, and samples are taken at various time points. The lipid fractions can be
extracted from the cells and separated and analyzed by LC-MS. From the LC-MS data, rates of
biodegradation (e.g., as t1/2 values) can be measured.
For example, the compound
O N Compound 1
includes an ester linkage in each aliphatic chain, which can undergo hydrolysis in a biological
environment, for example, when exposed to, e.g., a lipase or an esterase. The structure of the
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WO wo 2020/072324 PCT/US2019/053617 PCT/US2019/053617
compound, of course, influences the rate at which the compound undergoes biodegradation. Thus,
a related compound such as
N O COOEt O COOEt Compound 2
would be expected to exhibit a different rate of biodegradation. Greater effects on that rate would
be expected from changes in the structure of the compound at the site of hydrolysis. One
modification that can influence the rate of hydrolysis, and thereby influence the rate of
biodegradation and clearance from a subject's body, is to make the leaving group of the hydrolysis
reaction have a primary, rather than secondary, alcohol.
In one embodiment, a cationic lipid of any of the embodiments described herein has an in
vivo half life (t1/2) (e.g., in the liver, spleen or plasma) of less than about 3 hours, such as less than
about 2.5 hours, less than about 2 hours, less than about 1.5 hours, less than about 1 hour, less than
about 0.5 hour or less than about 0.25 hours. The cationic lipid preferably remains intact or has a
half-life sufficient to form a stable lipid nanoparticle which effectively delivers the desired active
pharmaceutical ingredient (e.g., a nucleic acid) to its target but thereafter rapidly degrades to
minimize any side effects to the subject. For instance, in mice, the cationic lipid preferably has a
t1/2 in the spleen of from about 1 to about 7 hours.
In another embodiment, a cationic lipid of any of the embodiments described herein
containing a biodegradable group or groups has an in vivo half life (t1/2) (e.g., in the liver, spleen
or plasma) of less than about 10% (e.g., less than about 7.5%, less than about 5%, less than about
2.5%) of that for the same cationic lipid without the biodegrable group or groups.
Some cationic lipids can be conveniently represented as a hydrophobic group combined
via a central moiety (such as a carbon atom) with a headgroup. By way of example, the compound:
O O O O N O O - 23
WO wo 2020/072324 PCT/US2019/053617
can be thought of as a combination of a headgroup, a central moiety, and two hydrophobic groups
as follows:
O Hydrophobic Groups new O O N /N Head Group K Central Moiety
Suitable cationic lipids include compounds composed of any combination of the head and
hydrophobic hydrophobic groups groups listed listed below below (in (in combination combination with with a a central central moiety moiety (such (such as as a a central central carbon carbon
atom).
Some suitable head groups include those depicted in Table 1A:
TABLE 1A
2020/07234 OM PCT/US2019/053617 LI9ES0/6I07SN/LOd OM
N O O O O N I-o |-o I-° 1-N I-° N O O O I-o I-° N ZI I-N ZI N
N 1-0 N N N
N O Ton- N N O Tom N O
O O O O N ZI NN ZI N N H N N H TN H 1-0-N= N 1-0 N 1-0 -O'n 1-ON-IN N N 1-0 \ NN N
1-0-N= N1-0- 1-0-N= 1-0-N N 1-0- -O-N= N N N N- N N- N -O-N= N1-0-1 N 1-0- -O-N= N1-0- -O-N=
N N-NI N- H N. N N \ Town N
\ N Tan Lan-Am N / N NN N N Tan- N Tan N- N N-N Tow N-N
WO wo 2020/072324 PCT/US2019/053617
N H2N HN N O (CH2)n O HN (CH) N (where n is 0-5) 5 N O 0 0 N * * N N O O O o 0 HN 0 HN (the carbon with an asterisk is O the tertiary carbon of the cation HN (the carbon with an asterisk is (CHn (CH) lipid and is not part of the head (where n is 0-5) the tertiary carbon of the cation group) lipid and is not part of the head
group) R R + + +/ - N N R N X X
- - X R = H, alkyl (e.g., R = H, alkyl (e.g., methyl) R = H, alkyl (e.g., methyl) methyl) X = halogen (e.g., Cl) C1) X = halogen (e.g., Cl) C1) X = halogen (e.g., Cl) C1)
HN (CH2)n (CH) (where n is 0-5)
Suitable primary groups include, but are not limited to, those that are a combination of a
head group from table 1A with a central carbon atom. Other suitable primary groups include
those in table 1B below:
Table 1B
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Oo N * * N 0
Some Some suitable suitable hydrophobic hydrophobic tail tail groups groups include include those those depicted depicted in in Table Table 2: 2:
TABLE 2
O O OJ O OJ R O R O O O O O O-R R O-R R O O R = Me, Et R = Me, Et
/ O R 01 R OR O O. O. O R R O O R = Me, Et R = Me, Et 23' O COOEt
n/v
WO wo 2020/072324 PCT/US2019/053617
O O MeO S F O O F o
O OMe S OMe OMe O O O O O OCF3 F OCF F O OMe O O O O O O S
O o
CF3 O O CF CF3 S CF
WO wo 2020/072324 PCT/US2019/053617
O F O F IZ N H mv F O O F IZ N O H www NVV OMe OMe o O O S ww SMe ww OMe O O S O MW SMe O O S O
m Cationic lipids include those having alternative fatty acid groups and other dialkylamino
groups, including those in which the alkyl substituents are different (e.g., N-ethyl-N-methylamino-, N-propyl-N-ethylamino- and the like). For those embodiments in which
R R¹Superscript(1) and R² areand R 2 are both longboth long chain chain alkyl, alkyl, alkenyl, alkynyl, alkenyl, alkynyl, or cycloalkylalkyl groups, they or cycloalkylalkyl can be the groups, they can be the
same or different. In general, lipids (e.g., a cationic lipid) having less-saturated acyl chains are
more easily sized, particularly when the complexes are sized below about 0.3 microns, for purposes
of filter sterilization. Cationic lipids containing unsaturated fatty acids with carbon chain lengths
in in the the range rangeofof C10C to to C20 are typical. C are typical. Other Otherscaffolds can can scaffolds also also be used be to separate used the amino to separate group the amino group - 29
WO wo 2020/072324 PCT/US2019/053617 PCT/US2019/053617
(e.g., the amino group of the cationic lipid) and the fatty acid or fatty alkyl portion of the cationic
lipid. Suitable scaffolds are known to those of skill in the art.
In certain embodiments, cationic lipids have at least one protonatable or deprotonatable
group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4),
and neutral at a second pH, preferably at or above physiological pH. Such lipids are also referred
to as cationic lipids. It will, of course, be understood that the addition or removal of protons as a
function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers
to the nature of the predominant species and does not require that all of the lipid be present in the
charged or neutral form. The lipids can have more than one protonatable or deprotonatable group,
or can be zwiterrionic.
In certain embodiments, protonatable lipids (i.e., cationic lipids) have a pKa of the
protonatable group in the range of about 4 to about 11. Typically, lipids will have a pKa of about
4 to about 7, e.g., between about 5 and 7, such as between about 5.5 and 6.8, when incorporated
into into lipid lipid particles. particles. Such Such lipids lipids will will be be cationic cationic at at aa lower lower pH pH formulation formulation stage, stage, while while particles particles
will be largely (though not completely) surface neutralized at physiological pH around pH 7.4.
One of the benefits of a pKa in the range of between about 4 and 7 is that at least some nucleic acid
associated with the outside surface of the particle will lose its electrostatic interaction at at
physiological pH and be removed by simple dialysis; thus greatly reducing the particle's
susceptibility to clearance. pKa measurements of lipids within lipid particles can be performed, for
example, by using the fluorescent probe 2-(p-toluidino)-6-napthalene sulfonic acid (TNS), using
methods described in Cullis et al., (1986) Chem Phys Lipids 40, 127-144, which is incorporated
by reference in its entirety.
In particular embodiments, the lipids are charged lipids. As used herein, the term "charged
lipid" is meant to include those lipids having one or two fatty acyl or fatty alkyl chains and a
quaternary amino head group. The quaternary amine carries a permanent positive charge. The head
group can optionally include a ionizable group, such as a primary, secondary, or tertiary amine
that may be protonated at physiological pH. The presence of the quaternary amine can alter the
pKa of the ionizable group relative to the pKa of the group in a structurally similar compound that
lacks the quaternary amine (e.g., the quaternary amine is replaced by a tertiary amine) In some
- 30 embodiments, a charged lipid is referred to as an "amino lipid." See, for example, provisional U.S.
patent application 61/267,419, filed December 7, 2009, which is incorporated by reference in its
entirety.
In one embodiment, the cationic lipid is a compound of formula (I), which has a branched
alkyl at the alpha position adjacent to the biodegradable group (between the biodegradable group
and the teriary carbon):
H R2 R² R¹. M1 R R X M R' NR 1 QN R b* X a b Z¹ Z1 R'
R2 R² M² Y z2
The Z²
H R2 R²
Formula (I)
or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein
R' is absent, hydrogen, or alkyl (e.g., C1-C4 alkyl); C-C alkyl);
with withrespect to R Superscript(1) respect to R¹ and R², and R2,
(i) (i) R1 R¹ and and R2 R² are are each, each, independently, independently, optionally optionally substituted substituted alkyl, alkyl, alkenyl, alkenyl, alkynyl, alkynyl,
cycloalkylalkyl, cycloalkylalkyl, heterocycle, or R10: heterocycle, or R¹;
(ii) (ii) RR¹ Superscript(1) and R2, with and R², together together the with the nitrogen nitrogen atom toatom to which which theythey are are attached,form attached, form an an
optionally substituted heterocylic ring; or
(iii) (iii) one oneofof R Superscript(1) R¹ and R² is and R2 is optionally optionally substituted substituted alkyl,alkyl, alkenyl, alkenyl, alkynyl,cycloalkyl, alkynyl, cycloalkyl,
cycloalkylalkyl, or heterocycle, and the other forms a 4-10 member heterocyclic ring or heteroaryl
(e.g., a 6-member ring) with (a) the adjacent nitrogen atom and (b) the (R)a group adjacent to the
nitrogen atom;
each occurrence of R is, independently, -(CR3R)-; -(CR³R)-; wo 2020/072324 WO PCT/US2019/053617 PCT/US2019/053617 each each occurrence occurrenceof of R³ R³ and and R4 are, independently R are, H, halogen, independently OH, alkyl, H, halogen, OH,alkoxy, alkyl,-NH2, R 10,-NH, R¹, alkoxy, alkylamino, or dialkylamino (in one preferred embodiment, each occurrence of R3 R³ and R4 are, R are, independently H or C1-C4 alkyl); C-C alkyl); each occurrence of R10 is independently R¹ is independently selected selected from from PEG PEG and and polymers polymers based based on on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized by n subunits,
(iii) n is a number-averaged degree of polymerization between 10 and 200 units, and (iv) wherein
the compound of formula has at most two R10 groups (preferably R¹ groups (preferably at at most most one one R¹ R10 group); group);
the dashed line to Q is absent or a bond;
when the dashed line to Q is absent then Q is absent or is -O-, -0-, -NH-, -S-, -C(O)-, -C(O)O- -C(0)0-
, -OC(O)-, -OC(O)-, -C(O)N(R4)-, -C(O)N(R)-,-N(R5)(())-, -N(R)C(O)-,-S-S-, -OC(O)O-, -S-S-, -O-N=C(R5)-, -0C(0)0-, -C(R5)=N-O-, -O-N=C(R)-, -C(R)=N-O-, - - OC(O)N(R5)-,-N(R5)C(O)N(R5)-,-N(R5)C(O)O-,-C(O)S-,-C(S)O- or -C(R5)=N-O-C(O)-; OC(O)N(R)-, -N(R°)C(O)N(R³)-, -N(R°)C(O)O-, -C(O)S-, -C(S)O- or or or -C(R³)=N-O-C(O)-;
when the dashed line to Q is a bond then (i) b is 0 and (ii) Q and the tertiary carbon adjacent
to it (C*) form a substituted or unsubstituted, mono- or bi-cyclic heterocyclic group having from
5 to 10 ring atoms (e.g., the heteroatoms in the heterocyclic group are selected from O and S,
preferably O);
each each occurrence occurrenceof of R5 R is, independently, is, H or H independently, alkyl (e.g. C1-C4 or alkyl (e.g.alkyl); C-C alkyl);
X X and and YYare areeach, independently, each, alkylene independently, or alkenylene alkylene (e.g., C4 or alkenylene to C20 Calkylene (e.g., or C4 to or C to to C alkylene
C20 alkenylene); C alkenylene);
M M¹¹and andM² M2are areeach, each,independently, independently,aabiodegradable biodegradablegroup group(e.g., (e.g.,-OC(O)-, -OC(O)-,-C(0)0-, -C(O)O-,- -
SC(O)-, SC(O)-, -C(O)S-, -C(O)S-,-OC(S)-, -C(S)O-, -OC(S)-, -S-S-, -C(S)O-, -C(R5)=N-, -S-S-, -N=C(R5)-, -C(R)=N-, -C(R5)=N-O-, -N=C(R)-, -O-N=C(R5)-, -C(R)=N-O-, -O-N=C(R)-,
-C(O)(NR5)-, -C(O)(NR)-, -N(R5)((()), -N(R)C(O)-, -C(S)(NR5)-, -C(S)(NR)-,-N(R5)C(O)-, -N(R)C(O)-,-N(R5)C(O)N(R5)-, -N(R°)C(O)N(R)-, -OC(O)O-, -0C(0)0-, - -
O-R1-1 O-R¹¹ NVV
OSi(R)O-, -C(O)(CR³R)C(O)O-, -OC(O)(CR³R)C(O)-, or O (wherein (whereinR R¹¹ 11 is isa aC2- C-
C8 alkyl or C alkyl or alkenyl)); alkenyl));
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WO wo 2020/072324 PCT/US2019/053617
each occurrence of R2 R² is, independently, C1-Cg alkyl C-C alkyl (e.g., (e.g., methyl, methyl, ethyl, ethyl, isopropyl, isopropyl, n-butyl, n-butyl,
in-pentyl, orn-hexyl); n-pentyl, or in-hexyl);
a is 1, 2, 3, 4, 5 or 6;
b is 0, O, 1, 2, or 3; and
Z Z¹¹ and and Z² Z² are are each, each,independently, C8-C14 independently, C-C alkyl alkyloror C8-C14 alkenyl, wherein C-C alkenyl, whereinthe alkenyl the alkenyl
group may optionally be substituted with one or two fluorine atoms at the alpha position to a double
my bond which is between the double bond and the terminus of Z1 Z¹ or Z2 Z² (e.g., ).
The R'R'R'N-(R)A-Q-(R)6 R'R¹R²N-(R)a-Q-(R)- group can be any of the head groups described herein,
including those shown in Table 1 below, and salts thereof. In one preferred embodiment,
R'R¹R²N-(R)a-Q-(R)- is is (CH)N-(CH)-C(O)O-, (CH3)2N-(CH2)3-C(O)O-, (CH)N-(CH)-NH-C(O)O-, (CH3)2N-(CH2)2-NH-C(O)O- (CH)N- (CH3)2N-
(CH2)2-OC(O)-NH-, or (CH)-OC(O)-NH-, or (CH3)2N-(CH2)3-C(CH3)=N-O- (CH)N-(CH)-C(CH)=N-O-.
In one embodiment, R1 R¹ and R2 R² are both alkyl (e.g., methyl).
In a further embodiment, a is 3. In another embodiment, b is 0.
In a further embodiment, a is 3, b is 0 and R is -CH2-. In yet -CH-. In yet aa further further embodiment, embodiment, aa is is 3, 3,
b b is is 0, 0,R Risis-CH2- -CH-and Q is and -C(O)O-. Q is In another -C(0)0-. embodiment, In another R Superscript(1) embodiment, R¹ and R²and R2 methyl, are are methyl, a is a is 3, 3, b b isis0,0,
R is -CH2- and QQ is -CH- and is -C(0)0-. -C(O)O-.
In another embodiment, X and Y are each, independently -(CH2)n- whereinnnis -(CH)n- wherein is44to to20, 20,
e.g., 4 to 18, 4 to 16, or 4 to 12. In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one exemplary
embodiment, X and Y are -(CH2)6-. -(CH). In In another another embodiment, embodiment, X and X and Y are Y are -(CH2)7-. -(CH)-. In another In yet yet another
embodiment, X and Y are -(CH2)9-. -(CH)-. InIn yet yet another another embodiment, embodiment, X X and and Y Y are are -(CH2)8-. -(CH)-.
In further embodiments, M M¹¹ and and M² M2 are are each, each, independently, independently, -OC(O)- -OC(O)- or or -C(0)0-. -C(O)O-. For For
example, in one embodiment, M M¹¹and andM² M²are areeach each-C(0)0-. -C(O)O-.
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PCT/US2019/053617
In one embodiment, the cationic lipid is a compound of formula (V), which has an alkoxy
or thioalkoxy (i.e., -S-alkyl) group substitution on at least one tail:
Z¹ R RR RR * X M ¹ N a b b M R'
R2 R² Y. Z² Y M²
Formula Formula (V) (V)
or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein
R', R 1, R², R¹, R2, R, R, R³, R3, R, R4, R 10, R¹, Q, M¹, Q, R, R5, M², M1, a, M², a, band and areb defined are defined as inas in formula formula (I); (I);
X and Y are each, independently, alkylene (e.g., C6-C8 alkylene) C-C alkylene) oror alkenylene, alkenylene, wherein wherein
the alkylene or alkenylene group is optionally substituted with one or two fluorine atoms at the
Z¹
a M alpha position to the M M¹¹ or or M² M2 group group (e.g., (e.g., );
Z Superscript(1) and Z² are each, independently, C8-C14 alkyl or C8-C14 alkenyl, wherein (i) the C8-C14 Z¹ and Z² are each, independently, C-C alkyl or C-C alkenyl, wherein (i) the C-C
alkyl alkyl or orC8-C14 alkenyl of C-C alkenyl of at at least leastone of of one Z1 Z¹ and and Z² is Z²substituted by one by is substituted or one more or alkoxy more(e.g., alkoxya (e.g., a
C1-C4 alkoxy C-C alkoxy such such asas -OCH3) -OCH) or or thioalkoxy thioalkoxy (e.g., (e.g., a C1-C4 a C-C thioalkoxy thioalkoxy such such as -SCH3) as -SCH) groups, groups, and and
(ii) the alkenyl group may optionally be substituted with one or two fluorine atoms at the alpha
position to a double bond which is between the double bond and the terminus of Z Z¹¹or orZ² Z2(e.g., (e.g.,
my
Z¹¹ and/or In one embodiment, the alkoxy substitution on Z and/or Z² Z2 is is at at the the beta beta position position from from the the
M M¹¹and/or and/orM² M2group. group.
In another embodiment, X and Y are each, independently -(CH2)n -(CH)n- wherein n is 4 to 20,
e.g., 4 to 18, 4 to 16, or 4 to 12. In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one exemplary
- 34 wo 2020/072324 WO PCT/US2019/053617 embodiment, X and Y are -(CH2)6-. -(CH). In In another another embodiment, embodiment, X and X and Y are Y are -(CH2)7-. -(CH)-. In another In yet yet another embodiment, X and Y are -(CH2)9-. -(CH)-. InIn yet yet another another embodiment, embodiment, X X and and Y Y are are -(CH2)8-. -(CH)-.
The R'R¹R²N-(R)a-Q-(R)- The group can be any group ofcanthe be any headof groups the head groups described described herein, herein, including those shown in Table 1 below, and salts thereof. In one preferred embodiment,
R'R¹R²N-(R)a-Q-(R)- is is (CH)N-(CH)-C(O)O-, (CH3)2N-(CH2)3-C(O)O-, (CH)N-(CH)-NH-C(O)O-, (CH3)2N-(CH2)2-NH-C(O)O-, (CH)N- (CH3)2N-
(CH2)2-OC(O)-NH-, or (CH)-OC(O)-NH-, or (CH)N-(CH)-C(CH)=N-O-. (CH3)2N-(CH2)3-C(CH3)=N-O-
In one embodiment, the cationic lipid is a compound of formula (VIA), which has one or
more fluoro substituents on at least one tail at a position that is either alpha to a double bond or
alpha to a biodegradable group:
R1 R¹ (R) (R) R' N Q * R R² R2 10 R10 R Formula (VIA)
or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein
R 1,R², R¹, R2,R, R,a, a,and andbbare areas asdefined definedwith withrespect respectto toformula formula(I); (I);
-0-, -NH-, -S-, -C(O)-, -C(O)O-, Q is absent or is -O-, -C(0)0-, -OC(O)-, -C(O)N(R4)-, -C(O)N(R)-, -N(R)C(O)-, - - -N(R5)(()),,
-OC(0)0-, -O-N=C(R)-, S-S-, .-OC(O)O-, -C(R)=N-O-, -O-N=C(R5)-, -OC(O)N(R)-, -C(R5)=N-O-, -N(R°)C(O)N(R³)-, -OC(O)N(R³)., -N(R°)C(O)O-, -N(R)C(O)N(R)-, -N(R3)(())-,
-C(O)S-, -C(S)O- or -C(O)S-,-C(S)O- or-C(R5)=N-O-C(O)-; -C(R²)=N-O-C(O)-;
R' is absent, hydrogen, or alkyl (e.g., C1-C4 alkyl); C-C alkyl); and and
each each of of R9 R and and R10 R¹ are are independently independentlyC12-C24 alkyl (e.g., C-C alkyl (e.g., C12-C20 alkyl), C-C alkyl), C12-C24 C-C alkenyl alkenyl
(e.g., (e.g.,C12-C20 alkenyl), ororC12-C24 C-C alkenyl), alkoxy (e.g., C-C alkoxy (e.g.,C12-C20 alkoxy) (a) C-C alkoxy) having (a) one or having onemore or more biodegradable groups and (b) optionally substituted with one or more fluorine atoms at a position
which is (i) alpha to a biodegradable group and between the biodegradable group and the tertiary
carbon atom marked with an asterisk (*), or (ii) alpha to a carbon-carbon double bond and between
the double bond and the terminus of the R9 orR¹ R or R10 group; group; each each biodegradable biodegradable group group independently independently
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WO wo 2020/072324 PCT/US2019/053617
interrupts the interrupts C12-C24 the alkyl, alkenyl, C-C alkyl, alkenyl, or oralkoxy alkoxygroup or is group or substituted at theat is substituted terminus of the C12- the terminus of the C-
C24alkyl, C alkyl, alkenyl, alkenyl, or or alkoxy alkoxygroup, group,
wherein (i) at at least leastone oneofof R9 Rand R10R¹contains and a fluoro contains group; a fluoro group;
(ii) the compound does not contain the following moiety:
O mm O
is an wherein is an optional bond; optional bond;andand (iii) the terminus of R9 and RR 10 R and ¹ 10 is is separated separated from from thethe tertiary tertiary carbon carbon atom atom marked marked
with an asterisk (*) by a chain of 8 or more atoms (e.g., 12 or 14 or more atoms).
In one preferred embodiment, the terminus of R9 and R¹ R and R10 isis separated separated from from the the tertiary tertiary
carbon atom marked with an asterisk (*) by a chain of 18-22 carbon atoms (e.g., 18-20 carbon
atoms).
In another embodiment, the terminus of the R9 and/or R¹ R and/or R10 has has the the formula formula -C(O)O-CF3. -C(O)O-CF.
In another embodiment, the cationic lipid is a compound of formula (VIB), which has one
or more fluoro substituents on at least one tail at a position that is either alpha to a double bond or
alpha to a biodegradable group:
R Superscript(1)
R¹. Z¹ R R X R R *
R' N b * X N a Q b M1 M
R² R2 Z² Y M²
Formula (VIB) wo 2020/072324 WO PCT/US2019/053617 or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein
R', , R1, R¹, R2, R², R,R, R3, R³, R,R4, R¹,R Q, 10, R,Q, R5, M¹, M1, M², a,M², anda, b and are b are defined defined as in as in formula formula (I); (I);
X and Y are each, independently, alkylene (e.g., C6-C8 alkylene) C-C alkylene) oror alkenylene, alkenylene, wherein wherein
the alkylene or alkenylene group is optionally substituted with one or two fluorine atoms at the
Z¹
a M alpha position to the M1 M¹ or M2 M² group (e.g., ); and and
Z¹ Z¹ and and Z2 Z²are areeach, independently, each, C8-C14 independently, C-Calkyl or or alkyl C8-C14 C-C alkenyl, alkenyl,wherein said wherein C8-C14 said C-C
alkenyl is optionally substituted by one or more fluorine atoms at a position that is alpha to a F, F. FF S my double bond (e.g., ),
wherein at least one of X, Y, Z , and Z² contains a fluorine atom. Z¹,
In In one one embodiment, embodiment,at at least one of least oneZ of Superscript(1) Z¹ and Z² isandsubstituted Z2 is substituted by fluoro by two two fluoro groups groups at ata a
position that is either alpha to a double bond or alpha to a biodegradable group. In one embodiment,
at at least leastone oneofof Z ¹Z¹and Z² Z² and hashas a terminal -CF3 -CF a terminal groupgroup at a position that is that at a position alpha is to alpha a biodegradable to a biodegradable
group (i.e., at least one of Z1 Z¹ and Z2 Z² terminates with an -C(O)OCF3 group). -C(O)OCF group).
For example, at least one of Z¹ and Z2 Z² may include one or more of the following moieties:
F. F F CF3 CF O O CF3 CF CF3 F F CF O F F F F F F O O
In one embodiment, X and Y are each, independently -(CH2)n- wherein -(CH)- wherein n n isis 4 4 toto 20, 20, e.g., e.g., 4 4
to 18, 4 to 16, or 4 to 12. In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one exemplary
embodiment, X and Y are -(CH2)7-. -(CH)-. InIn another another exemplary exemplary embodiment, embodiment, X X and and Y Y are are -(CH2)9-. -(CH)-. In In
yet another embodiment, X and Y are -(CH2)8-. -(CH)-.
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WO wo 2020/072324 PCT/US2019/053617
The R'R¹R²N-(R)a-Q-(R)- group can be any of the head groups described herein, The group can be any of the head groups described herein, including those shown in Table 1 below, and salts thereof. In one preferred embodiment,
is is R'R¹R²N-(R)a-Q-(R)- (CH3)2N-(CH2)3-C(O)O-, (CH)N-(CH)-C(O)O-, (CH3)2N-(CH2)2-NH-C(O)O-, (CH)N-(CH)-NH-C(O)O-, (CH3)2N- (CH)N- (CH2)2-OC(O)-NH-, oror (CH)-OC(O)-NH-, (CH3)2N-(CH2)3-C(CH3)=N-O-. (CH)N-(CH)-C(CH)=N-O-
In one embodiment, the cationic lipid is a compound of formula (VII), which has an acetal
group as a biodegradable group in at least one tail:
R¹. Z¹ R R X * N a b Q M
James R'
R2 R²
Formula (VII) Y M² Z²
or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein
R', , R 1, R¹, R²,R2, R, R, R³,R3, R, R4, R¹, R Q,10, R, Q, a, R5, and a, and defined b are b are defined as in formula as in formula (I); (I);
X and Y are each, independently, alkylene (e.g., C6-C8 alkylene) C-C alkylene) oror alkenylene, alkenylene, wherein wherein
the alkylene or alkenylene group is optionally substituted with one or two fluorine atoms at the
Z¹
alpha position to the M M¹¹or orM² M2group group(e.g., (e.g., M ); 2
M ¹and M¹ andM² M2are areeach, each,independently, independently,aabiodegradable biodegradablegroup group(e.g., (e.g.,-OC(O)-, -OC(O)-,-C(0)0-, -C(O)O-,--
SC(O)-, SC(O)-, -C(O)S-, -C(O)S-,-OC(S)-, -C(S)O-, -OC(S)-, -S-S-, -C(S)O-, -C(R5)=N-, -S-S-, -N=C(R5)-, -C(R)=N-, -C(R5)=N-O-, -N=C(R)-, -O-N=C(R5)-, -C(R)=N-O-, -O-N=C(R)-,
-C(O)(NR5)-, -C(O)(NR)-, -N(R5)((()),, -C(S)(NR5)-, -N(R5)((C)-, -N(R)C(O)-, -C(S)(NR)-, -N(R)C(O)-, -N(R5)C(O)N(R5)-, -N(R°)C(O)N(R)-,-OC(O)O-, -0C(0)0-, - -
O-R¹¹ s
OSi(R)O-, -C(O)(CR³R)C(O)O-, -OC(O)(CR³R)C(O)-, or O (wherein (whereinR R¹¹ 11 is isa aC4- C-
C10 alkyl or C alkyl or C4-C1o alkenyl)) C-C alkenyl));
with the proviso that at least one of M M¹¹ and and M² M2 is is O ; and ; and
Z¹ and Z2 Z² are each, independently, C4-C14 alkyl or C4-C14 alkenyl, wherein the alkenyl
group may optionally be substituted with one or two fluorine atoms at the alpha position to a double
bond bondwhich is between which the double is between thebond and the double terminus bond and ofthe Z Superscript(1) terminus ofor Z¹ Z² (e.g., or Z² (e.g., ).
O-R¹¹ O-R me
In one embodiment, each of M M¹¹ and and M² M2 is is O
In another embodiment, X and Y are each, independently -(CH2)n- wherein nn is -(CH)n- wherein is 44 to to 20, 20,
e.g., 4 to 18, 4 to 16, or 4 to 12. In one embodiment, n is 4, 5, 6, 7, 8, 9, or 10. In one exemplary
embodiment, X and Y are -(CH2)6-. -(CH). In In another another embodiment, embodiment, X and X and Y are Y are -(CH2)7-. -(CH)-. Inanother In yet yet another
embodiment, X and Y are -(CH2)9-. -(CH)-. InIn yet yet another another embodiment, embodiment, X X and and Y Y are are -(CH2)- -(CH)-.
The R'R¹R²N-(R)a-Q-(R)- group can be any of the head groups described herein, The group can be any of the head groups described herein, including those shown in Table 1 below, and salts thereof. In one preferred embodiment,
R'R¹R²N-(R)a-Q-(R)- is is (CH)N-(CH)-C(O)O-, (CH3)2N-(CH2)3-C(O)O-, (CH)N-(CH)-NH-C(O)O-, (CH3)2N-(CH2)2-NH-C(O)O-, (CH)N- (CH3)2N-
(CH2)2-OC(O)-NH-, or (CH)-OC(O)-NH-, or (CH3)2N-(CH2)3-C(CH3)=N-O- (CH)N-(CH)-C(CH)=N-O-.
In another embodiment, the cationic lipid or a salt thereof has: (i) a central carbon atom,
(ii) a nitrogen containing head group directly bound to the central carbon atom, and
(iii) two hydrophobic tails directly bound to the central carbon atom, wherein each
hydrophobic tail is of the formula -Re-M-R -R°-M-Rfwhere whereRe Reis isaaC4-C14 C4-C14alkyl alkylor oralkenyl, alkenyl,MMis isaa
biodegradable biodegradablegroup, andand group, Rf is Rf ais branched alkyl or a branched alkenyl alkyl (e.g., a (e.g., or alkenyl C10-C20 aalkyl C-C or C10-C20 alkyl alkenyl), or C-C alkenyl),
such that (i) the chain length of -Re-M-R4 -R°-M-Rf is at most 20 atoms (i.e. the total length of the tail from
the first carbon atom after the central carbon atom to a terminus of the tail is at most 20), and (ii)
the group -Re-M-R -R°-M-Rfhas hasat atleast least20 20carbon carbonatoms atoms(e.g., (e.g.,at atleast least21 21atoms). atoms).Optionally, Optionally,the thealkyl alkylor or
- 39 wo 2020/072324 WO PCT/US2019/053617 alkenyl group in Re may be substituted with one or two fluorine atoms at the alpha position to the
Rf
a M M M¹¹ or or M² M2 group group (e.g., (e.g., ). Also, optionally, the alkenyl group in R Rff may may be be
substituted with one or two fluorine atoms at the alpha position to a double bond which is between
myS the double bond and the terminus of R Rff(e.g., (e.g., ). ).
In one embodiment, the cationic lipid (such as of formulas I-VII) has assymetrical
hydrophobic groups (i.e., the two hydrophobic groups have different chemical formulas). For
example, the cationic lipid can have the formula:
R12-M1-R13 R¹²-M-R¹³
Primary Group
Formula (VIII)
or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein
G G is is branched branchedoror unbranched C3-C15 unbranched C-Calkyl, alkenyl alkyl, or alkynyl alkenyl (e.g.,(e.g., or alkynyl a n-C8 aalkyl n-C n-C9 alkylalkyl, n-C alkyl,
or or n-C10 alkyl); n-C alkyl);
R 12is R¹² isaabranched branchedor orunbranched unbranchedalkylene alkyleneor oralkenylene alkenylene(e.g., (e.g.,C-C C6-C20 alkylene alkylene or C6-C20 or C-C
alkenylene alkenylenesuch suchas as C12-C20 alkylene or C-C alkylene or C12-C20 alkenylene); C-C alkenylene);
M1 isaabiodegradable M is biodegradablegroup group(e.g., (e.g.,-OC(O)-, -OC(O)-,-C(0)0-, -C(O)O-,-SC(O)-, -SC(O)-,-C(O)S-, -C(O)S-,-OC(S)-, -OC(S)-,-C(S)O- -C(S)O-
, -S-S-, -C(R5)=N-, -S-S-, -C(R)=N-, -N=C(R5)-, -N=C(R)-, -C(R5)=N-O-, -C(R)=N-O-,-O-N=C(R5)-, -O-N=C(R)-,-C(O)(NR5)-, -C(O)(NR)-,-N(R5)(()),, -N(R)C(O)-, --
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C(S)(NR5)-, -N(R5)((()), C(S)(NR)-, -N(R)C(O)-, N(R5)C(O)N(R5)- -OC(O)O-, -N(R°)C(O)N(R²)-, -0C(0)0-, -OSi(R5)20-, -C(0)(CR'R*)C(O)0-, -OSi(R)O-, -C(O)(CR³R)C(0)0-, -
O-R¹¹ mm
OC(O)(CR`R*)C(O)-,or OC(O)(CR³R)C(O)-, or O (wherein (whereinR R¹¹ 11 is isa aC2-C8 C-C alkyl alkylororalkenyl)); alkenyl));
R³ R³ and andR4R are are defined definedas as in in formula (I); (I); formula
each each occurrence occurrenceof of R5 R is, independently, is, H or H independently, alkyl (e.g., (e.g., or alkyl C1-C4 alkyl); C-C alkyl);
R 13 is R¹³ is branched branchedororunbranched C3-C15 unbranched C-C alkyl, alkyl,alkenyl or alkynyl; alkenyl and and or alkynyl;
Primary Group
comprises a protonatable group having a pKa of from about 4 to about 13,
more preferably from about 5 to about 8 (e.g. from about 5 to about 7, or from about 5 to about
6.5, or from about 5.5 to about 6.5, or from about 6 to about 6.5).
In one embodiment, the primary group includes (i) a head group, and (ii) a central moiety
(e.g., a central carbon atom) to which both the hydrophobic tails are directly bonded.
Representative central moieties include, but are not limited to, a central carbon atom, a central
nitrogen atom, a central carbocyclic group, a central aryl group, a central hetrocyclic group (e.g.,
central tetrahydrofuranyl group or central pyrrolidinyl group) and a central heteroaryl group.
Primary Group 6n in my N Representative 's include, but are not limited to, ;;
O * O N n N n n N N ;; ; N ; n n
O O N N N n N n N n N N $ n H ; H ;; H ;
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nov N S N n N n O N n N n N n. my ; ;; ; n ;
O S. Nm S in N S N n n and ; where n is 0-6.
Representative asymmetrical cationic lipids include:
O X O N W y
W y
N W y
wherein W is 0, 1, 2, or 3; and X and y are each independently 1, 2, 3, 4, 5, 6, or 7.
In one embodiment each R is, independently, -(CR3R)-, -(CR³R)-, wherein R3 R³ and R4 areeach, R are each,
independently, independently,H H or or alkyl (e.g., alkyl C1-C4 (e.g., alkyl). C-C For For alkyl). example, in oneinembodiment example, each R is, one embodiment each R is, independently, -(CHR4)-, whereineach -(CHR)-, wherein eachRR4 is, is, independently independently H H oror alkyl alkyl (e.g., (e.g., C1-C4 C-C alkyl). alkyl). In In
another another embodiment, embodiment,each R is, each independently, R is, -CH2-,-CH-, independently, -C(CH3)2- or -CH(iPr)- -C(CH)- (where (where or -CH(iPr)- iPr is iPr is
isopropyl). In another embodiment, each R is -CH2-. -CH-.
- 42 wo 2020/072324 WO PCT/US2019/053617
In another embodiment R5 is, in R is, in each each case, case, hydrogen hydrogen or or methyl. methyl. For For example, example, RR5 can can be, be,
in each case, hydrogen.
-C(0)0-, -OC(O)-,-C(O)N(R5)-,-N(R5)C(O)-,-S-S-, In one embodiment, Q is absent, -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, -S-S-, - -
OC(O)O-, -C(R5)=N-O-,-OC(O)N(R5)-,-N(R5)C(O)N(R5)-,-N(R5)C(O)O-, -C(O)S-,-C(S)O- or or
-C(R5)=N-O-C(0)-. In -C(R³)=N-O-C(O)-. In one one embodiment, embodiment, QQ is is-C(O)O-. -C(0)0-.
In one embodiment, the dashed line to Q is absent, b is 0 and R'R'R2N'(R)a-Q- R'R¹R²N-(R)a-Q- and the
tertiary carbon adjacent to it (C*) form the following group:
O * 3/3
n O N
where n is 1 to 4 (e.g., n is 2).
In one embodiment, the dashed line to Q is absent, b is 0 and R'R'R2N-(R)a-Q- R'R¹R²N-(R)a-Q- and the
tertiary carbon adjacent to it form the following group:
R¹ R1 O O * R' R' 3/2 N C (R)a O 0 R² n
where n is 1 to 4 (e.g., n is 2), and R 1,R², R¹, R2,R, R,a, a,and andbbare areas asdefined definedwith withrespect respectto toformula formula(I). (I).In In
one embodiment, a is 3.
In one embodiment, the dashed line to Q is absent, b is 0 and R'R'R2N-(R)a-Q R'R¹R²N-(R)a-Q-and andthe the
tertiary carbon adjacent to it form the following group:
R1 R¹ O R' *n N C /3/2 (R)a n O m R2 R² wo 2020/072324 WO PCT/US2019/053617 where n is 1 to 4 (e.g., n is 2), and R 1,R², R¹, R2,R, R,a, a,and andbbare areas asdefined definedwith withrespect respectto toformula formula(I). (I).In In one embodiment, a is 0. For example, the group can be:
R Superscript(1)
R¹ O O R' N C O 3/3
R2 (R)a (R) n O m R²
In one one embodiment, embodiment, b is b is 0. another 0. In In another embodiment, embodiment, a is 2,a3,isor2, 3, or 4 and 4 O. b is andFor b example, is 0. For example,
O, and Q is -C(O)O-. in one embodiment, a is 3 and b is 0. In another embodiment, a is 3, b is 0, -C(0)0-.
In certain embodiments, the biodegradable group present in the cationic lipid is selected
from an ester (e.g., -C(O)O- -C(0)0- or -OC(O)-), -0C(0)-), disulfide (-S-S-), oxime (e.g., -C(H)=N-O- or -O- -0-
N=C(H)-), -C(O)-O-, -OC(O)-, -C(R5)=N-, -N=C(R5)-, -C(R5)=N-O-, -O-N=C(R5)-, -O-C(O)O-, N=C(H)-), -C(0)-0-, -N=C(R)-, -O-N=C(R), -0-C(0)0-, -C(O)N(R5), -N(R5)(()),-C(S)(NR)-, -C(O)N(R), -N(R)C(O)-, -C(S)(NR5)-, (NR5)C(S)-, (NR)C(S)-, -N(R5)C(O)N(R5)-, -N(R²)C(O)N(R²)-, -C(O)S-, -C(O)S-, -SC(O)-, -SC(O)-, -
C(S)O-,-OC(S)-, -OSi(R5)20-, -C(0)(CR'R')C(O)0-, -OSi(R)O-, -C(O)(CR³R)C(O)O-, or or -OC(O)(CRR)C(O)-- -OC(O)(CR³R)C(O)-.
In a preferred embodiment of the aforementioned biodegradable cationic lipids, the
biodegradable cationic lipid has a logP value of at least 10.1 (as calculated by the software
available at http://www.molinspiration.com/services/logp.htm http://www.molinspiration.com/services/logp.htmlfrom fromMolinspiration Molinspiration
Cheminformatics of Slovensky Grob, Slovak Republic). More preferably, the logP value is at least
10.2 or 10.3.
In another preferred embodiment of the aforementioned biodegradable cationic lipids, the
biodegradable cationic lipid in the lipid nanoparticle has a HPLC retention time (relative to the
retention time of cholesterol in the lipid nanoparticle), hereafter referred to as tlipid - tchol, of at least
1.4. (The HPLC parameters are provided in the examples below. Unless otherwise specified, the
formulation of the lipid nanoparticle used is that described in Example 31). More preferably, the
tlipid - tchol value is at least 1.75, 2.0, or 2.25.
Yet another embodiment is a biodegradable cationic lipid having (i) a logP value of at least
10.1 and/or a tlipid - tchol, of at least 1.4, and (ii) one or more biodegradable groups (such as an ester
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group) located in the mid- or distal section of a lipidic moiety (e.g., a hydrophobic chain) of the
cationic lipid.
In a preferred embodiment, the cationic lipid includes a branched alkyl or branched alkenyl
group in its biodegradable group(s). In another preferred embodiment, the cationic lipd has a logP
of at least 10.2 or 10.3. In yet another preferred embodiment, the cationic lipid has a tlipid - tchol,
of at least 1.75, 2.0, or 2.25. The cationic lipid preferably has a pKa of from about 4 to about 7
(such as 6.0 to 6.5).
In one embodiment, the cationic lipid having a logP value of at least 10.1 and/or a tlipid -
tchol, of at least 1.4 comprises (a) a head group (preferably a nitrogen containing head group, such
as the head groups described herein), (b) at least two hydrophobic tails, each of the formula -
(hydrophobic chain)-(biodegradable group)-(hydrophobic chain), and (c) a linker group (for
instance, a single central carbon atom) which is bound to the head group and the hydrophobic tails.
The cationic lipid preferably has one, two, three, four or more of the properties listed below:
(i) a pKa of from about 4 to about 7 (such as 6.0 to 6.5);
(ii) in at least one hydrophobic tail (and preferably all hydrophobic tails), the
biodegradable group is separated from the terminus of the hydrophobic tail by from about 6 to
about 12 carbon atoms (for instance, 6 to 8 carbon atoms or 8 to 12 carbon atoms),
(iii) for at least one hydrophobic tail (and preferably all hydrophobic tails), the chain
length from the linker group to the terminus of the hydrophobic tail is at most 21 (e.g., at most 20,
or from about 17 to about 21, from about 18 to about 20, or from about 16 to about 18) (The
atom(s) in the linker group are not counted when calculating the chain length.);
(iv) (iv) for at least one hydrophobic tail (and preferably all hydrophobic tails), the total
number of carbon atoms in the hydrophobic tail is from about 17 to about 26 (such as from about
19 to about 26, or from about 21 to about 26);
(v) for at least one hydrophobic tail (and preferably all hydrophobic tails), the number
of carbon atoms between the linker group and the biodegradable group ranges from about 5 to
about 10 (for example, 6 to 10, or 7 to 9);
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(vi) for at least one hydrophobic tail (and preferably all hydrophobic tails), the total
number of carbon atoms between the linker group and the terminus of the hydrophobic tail is from
about 15 to about 20 (such as from 16 to 20, 16 to 18, or 18 to 20);
(vii) (vii) forat for at least least one one hydrophobic hydrophobictail (and(and tail preferably all hydrophobic preferably tails), tails), all hydrophobic the totalthe total
number of carbon atoms between the biodegradable group and the terminus of the hydrophobic
tail is from about 12 to about 18 (such as from 13 to 25);
(viii) for at least one hydrophobic tail (and preferably all hydrophobic tails), the terminal
hydrophobic chain in the hydrophobic tail is a branched alkyl or alkenyl group, for example, where
the branching occurs at the a, B,,Y, , ß, ororposition 8 position on on thethe hydrophobic hydrophobic chain chain relative relative to to thethe
biodegradable group;
(ix) when (ix) when formulated formulated as as a lipid a lipid nanoparticle nanoparticle (such (such as as in in Example Example 1),1), thethe cationic cationic lipid lipid
has an in vivo half life (t1/2) (t/) inin the the liver liver ofof less less than than about about 3 3 hours, hours, such such asas less less than than about about 2.5 2.5 hours, hours,
less than about 2 hours, less than about 1.5 hours, less than about 1 hour, less than about 0.5 hour
or less than about 0.25 hours;
(x) when formulated as a lipid nanoparticle (such as in Example 1), the cationic lipid
is eliminated from the liver in mice with a greater than 10-fold reduction in lipid levels relative to
Cmax withinthe C within thefirst first 24 24 hours hourspost-dose; post-dose;
(xi) (xi) when formulated as a lipid nanoparticle (such as in Example 1), the cationic lipid
is eliminated from the spleen in mice with an equal or greater than 10-fold reduction in lipid levels
relative relativetotoCmax within the C within the first first168 168hours post-dose; hours and and post-dose;
(xii) (xii) whenformulated when formulatedasasa alipid lipidnanoparticle nanoparticle(such (suchasasininExample Example1), 1),the thecationic cationiclipid lipid
is eliminated from plasma with a terminal plasma half-life (t1/2B) (t1/2ß) in rodents and non-human
primates of 48 hours or shorter.
Suitable cationic lipids include compounds having any combination of some or all of the
aforementioned properties. These properties provide a cationic lipid which remains intact until
delivery of an active agent, such as a nucleic acid, after which cleavage of the hydrophobic tail
occurs in vivo. For instance, the compounds can have all of properties (i) to (viii) (in addition to
the logP or tlipid - tchol value). In another embodiment, the compounds have properties (i), (ii), (iii),
and (viii). In yet another embodiment, the compounds have properties (i), (ii), (iii), (v), (vi), and
(viii).
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For cationic lipid compounds which contain an atom (e.g., a nitrogen atom) that carries a
positive charge, the compound also contains a negatively charged counter ion. The counterion can
be any anion, such as an organic or inorganic anion. Suitable examples of anions include, but are
not limited to, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate,
ascorbate, a-ketoglutarate, a-glycerophosphate, -ketoglutarate, -glycerophosphate, halide halide (e.g., (e.g., chloride), chloride), sulfate, sulfate, nitrate, nitrate, bicarbonate, bicarbonate,
and carbonate. In one embodiment, the counterion is a halide (e.g., Cl). C1).
Representative central moieties include, but are not limited to, a central carbon atom, a
central nitrogen atom, a central carbocyclic group, a central aryl group, a central hetrocyclic group
(e.g., central tetrahydrofuranyl group or central pyrrolidinyl group) and a central heteroaryl group.
Additionally, the central moiety can include, for example, a glyceride linker, an acyclic glyceride
analog linker, or a cyclic linker (including a spiro linker, a bicyclic linker, and a polycyclic linker).
The central moiety can include functional groups such as an ether, an ester, a phosphate, a
phosphonate, a phosphorothicate, phosphorothioate, a sulfonate, a disulfide, an acetal, a ketal, an imine, a hydrazone,
or an oxime. Other central moieties and functional groups are suitable as well.
In one embodiment, the cationic lipid is a racemic mixture. In another embodiment, the
cationic lipid is enriched in one diastereomer, e.g. the cationic lipid has at least 95%, at least 90%,
at least 80% or at least 70% diastereomeric excess. In yet another embodiment, the cationic lipid
is enriched in one enantiomer, e.g. the lipid has at least 95%, at least 90%, at least 80% or at least
70% enantiomer excess. In yet another embodiment, the cationic lipid is chirally pure, e.g. is a
single optical isomer. In yet another embodiment, the cationic lipid is enriched for one optical
isomer.
Where a double bond is present (e.g., a carbon-carbon double bond or carbon-nitrogen
double bond), there can be isomerism in the configuration about the double bond (i.e. cis/trans or
E/Z isomerism). Where the configuration of a double bond is illustrated in a chemical structure, it
is understood that the corresponding isomer can also be present. The amount of isomer present can
vary, depending on the relative stabilities of the isomers and the energy required to convert
between the isomers. Accordingly, some double bonds are, for practical purposes, present in only
a single configuration, whereas others (e.g., where the relative stabilities are similar and the energy
of conversion low) may be present as inseparable equilibrium mixture of configurations.
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PCT/US2019/053617
In some cases, a double-bonded unsaturation is replaced by a cyclic unsaturation. The
cyclic unsaturation can be a cycloaliphatic unsaturation, e.g., a cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl group. In some cases, the cyclic group can be
a polycyclic group, e.g., a bicyclic group or tricyclic group. A bicyclic group can be bridged, fused,
or have a spiro structure. In some cases, a double bond moiety can be replaced by a cyclopropyl
moiety, e.g., can be replaced by
Other suitable Other suitabletail groups tail includes groups thosethose includes of theofformula -R¹²-M¹-R¹³ the formula wherewhere R 12R¹² is is a a C4-C14 C4-C14
alkyl alkyl or orC4-C14 alkenyl, MM¹¹ is C-C alkenyl, is a a biodegradable biodegradable group as defined group above, as defined and R13 above, is R¹³ and a branched alkyl is a branched alkyl
or alkenyl or alkenyl(e.g., (e.g., a C-C alkyl a C10-C20 or C10-C20 alkyl or C-C alkenyl), alkenyl),such such that (i) the that (i) thechain chain length length of of -R¹²-M¹-R¹³
is at most 21 atoms (i.e., the total length of the tail from the first carbon after the tertiary carbon
(marked with (marked with an an asterisk) asterisk) to to a a terminus terminus of of the the tail tail is is at at most most 21), 21), and and (ii) (ii) the the group group -R¹²-M¹-R¹³
has at least 20 carbon atoms (e.g., at least 21 or 22 carbon atoms).
In one In one preferred preferredembodiment, the the embodiment, chain length chain of -R¹²-M¹-R¹³ length of is at is at most most 21 (e.g., 21 (e.g., at at most most
20). For example, the chain length can be from about 17 to about 24 or from about 18 to about 20.
In one In one embodiment, embodiment,thethe total carbon total atom atom carbon content of each content oftail each(-R¹²-M¹-R¹³) tail is fromis about from about
17 to about 26. For example, the total carbon atom content can be from about 19 to about 26 or
from about 21 to about 26.
In one embodiment, the tail has the formula:
O -R13 0-R¹³
where R13 R¹³ is an alkyl or alkenyl group having from about 13 to about 17 carbon atoms, and the
total carbon length of the tail from the first carbon (the leftmost carbon atom above) to a terminus
of the tail is at most 20. Preferably, the tail has from about 22 to about 26 carbon atoms. In one
embodiment, the maximum length of R 13from R¹³ fromits itsattachment attachmentpoint pointto tothe theester estergroup groupof ofthe the
compound is 12 carbon atoms (e.g., the maximum length can be 11 carbon atoms). In one preferred
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embodiment, the branch in the alkyl or alkenyl group is at the S-position or later -position or later from from the the point point of of
attachment of R 13 to R¹³ to the the ester ester group. group. Suitable Suitable R¹³ R 13 groups groups include, include, but but are are not not limited limited toto
C13 (C21) C14 (C22) C15 (C23) Length: C9 (18) Length: C9 (18) Length: C10 (19)
nw 23" m C13 (C21) C14 (C22) C15 (C23) Length: C9 (18) Length: C9 (18) Length: C10 (19)
C16 (C24) C17 (C25) Length: C10 (19) Length: C11 (20)
Mrs.
my
C16 (C24) C17 (C25) Length: C10 (19) Length: C11 (20)
C13 (C21) C15 (C23) Length: C8 (17) Length: C9 (18)
For example, the cationic lipid can be
O R¹³ o-R13 O O O * N R¹³ o-R¹3 O O
- 49 or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), where R13 R¹³ is selected from the groups mentioned above.
Another example Another example is is a tail a tail of formula of the the formula
O -R¹3 -R¹³
where R13 R¹³ is an alkyl or alkenyl group having from about 13 to about 15 carbon atoms, and the
total carbon length of the tail from the first carbon (i.e., the leftmost carbon atom, which is attached
to a tertiary carbon) to a terminus of the tail is at most 20. Preferably, the tail has from about 24
to about 26 carbon atoms. In one embodiment, the maximum length of R 13 from R¹³ from its its attachment attachment
point to the ester group of the compound is 10 carbon atoms (e.g., the maximum length can be 9
carbon atoms). In one preferred embodiment, the branch in the alkyl or alkenyl group is at the 8- -
position or later from the point of attachment of R13 R¹³ to the ester group. Suitable R13 R¹³ groups include,
but are not limited to
3/2
C13 (C23) C14 (C24) Length: C9 (20) Length: Length:C9C9(20) (20)
233
C13 (C23) C14 (C24) Length: C9 (20) Length: C9 (20)
C13 (C24) Length: C8 (19)
For example, the cationic lipid can be
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WO wo 2020/072324 PCT/US2019/053617
O R¹³ o-R13 O O O N * -R¹³ o-R13 O O
or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), where R13 R¹³ is selected from the
groups above.
The R 13 group R¹³ group may may be be derived derived from from aa natural natural product, product, such such as as dihydrocitgronellol, dihydrocitgronellol,
lavandulol, phytol, or dihydrophytol. In one embodiment, the R13 R¹³ group in the tails above is a
dihydrocitronellol group (either as a racemic group or a chirally pure group):
For example, the cationic lipid having a dihydroitronellol group can be
or
nwv
or a salt thereof (e.g., a pharmaceutically acceptable salt thereof).
In In another anotherembodiment, the the embodiment, R 13R¹³ group in the group in tails above is the tails a lavandulol above group or agroup is a lavandulol homolog or a homolog
of it as shown below:
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~~~~ Lavandulol homolog
In another embodiment, the R13 R¹³ group in the tails above is a phytol or dihydrophytol group:
Phytol
Dihydrophytol
For instance, the cationic lipid can be:
A cationic lipid of the fomula:
can also be thought of as a combination of a headgroup, a linker moiety, and two parts of the
hydrophobic chains as follows:
- 52 wo 2020/072324 WO PCT/US2019/053617
Hydrophobic chain I or la Hydrophobic chain II or lla O
2
ww Biocleavable moiety I or la
N in ww O 3 in
Hydrophobic chain II Head Head Group Group Linker Hydrophobic chain I
Biocleavable moiety I
Various headgroups, linker moieties, and hydrophobic chains I and II are listed below.
Suitable cationic lipids include compounds composed of any combination of the head, linker,
hydrophobic chain I, and hydrophobic chain II groups listed below.
Table 2A - Representative headgroups
new
nn ANN
N N- N N - - N N- Z N N- Z Z you me ANN now
N-½ HN N HN HN NI N N- N - JWV JVVV JWV will N nvv
N-3 N N HN N ZI
N nw Z nov my my N H
HN nim N N HN HN HN
+ + MVV
MVV N R-N R-N N O X1 N N N HN R = H, alkyl; X = halogen
in
+ + me
R-N R-N R-N N N N X1 ( X1 R == H, H,alkyl; X = Xhalogen alkyl; halogen R = H, alkyl; X = halogen my
Nin Nin N N my N mm my mm mr mm
NJ N 3r 25 N N N3, 3'3'
N3' 3.3
NH NH N HN H2N IZ N H2N NH IZ HN N N 323'3' HN-(CH2)n HN-(CH)- HN N H N 3.22 3'3' H (where n is 0-5)
\ N num N HN HN N N N N N N Itn N N I - n N- n = 0-6
+ ^^^^
Z ~~~~ N
^^^^ /
3'3" /
in 33 3'3" O
N N N N N Nm N z N I N N
Table 2B - Representative linker groups
s in JWV O n 23 O in m O n 2
n m m m mv n O MV MM m ==0-5,n 0-5, n == 0-3 0-3 n = 0-5 m = 0-5; n = 0-3 m = 1-5; n = 0-3
you JVVV in S O mv mw 23 m O n n O MW mr 3 my n 33 m = 0-5; n = 0-3 n 2 n = 0-3 n=0-3 n = 0-3 n=0-3
( X O O in Inn in IZ N w HollyHy N X Im n n N HYX m o 0 H m H mH n
m = 0-5; n = 0-3 m=0-5;n=0-3 m ==0-5;n = 0-5; n = 0-3 0-3 m m == 1-4; 1-4;n/o = 0-3 n/o = 0-3 m = 0-5; n = 0-3 m=0-5;n=0-3 x = O or S x=OrS in mv 133 S S n my n NN 3 N
m = 0-5; n = 0-3 m=0-5;n=0-3 N H n
the m m ==0-5;n S
0-5; n == 0-3 0-3 n m mm == 0-5; 0-5; nn == 0-3 0-3 n R n = 0-5
O O in O 3~ JVVV
n we N n
the /m m 33 n mR o O O MV m = 1-4; n = 0-3 m = 1-4; n/o = 1-3 n = 1-5 n=1-5 R = COOH, COOMe, COOEt, CN, CONH2 CONHMe R R1 N R R N O nnn
rivv -No n o- n O n O 13 2 nn o- n =0-5 n 0-5 3n 0-5n = 0-5 n = 0-6 n ==0-6 0-6
3 RR=H,Me, = H, Me,Et, Et, Pr, Pr, allyl allyl R=Me, R = Me,Et, Et, Pr, allyl Pr, allyl R1=Me, Et, Pr, R1= Me, Et, Pr,allyl allyl
O sur
S IZ n n in N N n mH mH H n = 0-6
m = 0-5; n == 0-3 m=0-5;n 0-3
Table 2C - Representative hydrophobic chain I and/or Ia, and combination thereof
in you p p=0-15 = p 0-15 p = 0-15, 0-15,q == 0-15 0-15
and /q
p == 0-15, 0-15,q q=0-15 = 0-15 p = 0-15, q = 1-4, r = 0-15 0-15,q=1-4,r=0-15
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WO wo 2020/072324 PCT/US2019/053617
in
p==0-15,q = 0-15, q == 1-4, 1-4, rr ==0-15 0-15
3/2 p 23 p q 0-15,qq==0-6 p = 0-15, 0-6 OMe p==0-15 = 0-15
R n
my n m n = 1-7 n=1-7 m = 0-4; n = 0-4; R = Me, Et, Pr, iPr, Bu, iBu
R PL PA m Jn q m = 1-4, n = 1-10, p = 0-15, q=0-15 q = 0-15 R = Me, Et, OMe
Table 2D - Representative biodegradable moieties I and/or la Ia and combinations thereof
O 13" O O O O S S 3 NVV mm NWV
O nn O R O ^^^^
--O-N N 3,5 Omm 0-2 O O O O O O O O ( 2 nn O nov
O n = 0-6 =0-6
O O O S O S O O O O mS O O R R X N-o-r JUNE
N { - O O O- my R = H, Me, Et, cyclic alkyl, alicylic, aromatic O.SS X = CH, O.
56 -- --
IIa and combinations thereof Table 2E - Representative hydrophobic chain II and/or Ila
n 7m m in n = 0-6; m = 0-16 n
/ n = 0-6
33' in
R /n n /n n my m /n n = 0-8 n = 0-8; m = 0-6 n n = 0-8 n=0-8 R =OMe, Me, Et, n-Pr, n-Bu
R R 3/2 my IYnn /n in /n my n p n = 0-8 n = 0-8 mm R =OMe, Me, Et, Pr R =OMe, Me, Et, Pr m =0-6; n = 0-6; p = 0-6
In \n In Yn Yn m Vn 2 2 m p p q Mm I p
m =0-6; n = 0-6; p =0-6 = 0-6 m =0-6; n=0-6;p n = 0-6;= p= =0-6 0-6 m =0-6; n = 0-6; p = 0-6; q = 0-6
In one embodiment, the cationic lipid is the following compound, and salts thereof
(including pharmaceutically acceptable salts thereof). This cationic lipid is suitable for forming
nucleic acid-lipid particles.
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WO wo 2020/072324 PCT/US2019/053617
N o
O 0
N X Alternatively, for the compounds above having a head of the formula
(where X can be, for example, -C(O)O-), the head can have one methylene unit between the X
group (or other functional group) and nitrogen atom. For example, the head can be:
N X Cationic lipids include those having alternative fatty acid groups and other dialkylamino
groups than those shown, including those in which the alkyl substituents are different (e.g.,
N-ethyl-N-methylamino-, and N-propyl-N-ethylamino-).
Included in the instant invention is the free form of the cationic lipids described herein, as
well as pharmaceutically acceptable salts and stereoisomers thereof. The cationic lipid can be a
protonated salt of the amine cationic lipid. The term "free form" refers to the amine cationic lipids
in non-salt form. The free form may be regenerated by treating the salt with a suitable dilute
aqueous base solution such as dilute aqueous NaOH, potassium carbonate, ammonia and sodium
bicarbonate.
The pharmaceutically acceptable salts of the cationic lipids can be synthesized from the
cationic lipids which contain a basic or acidic moiety by conventional chemical methods.
Generally, the salts of the basic cationic lipids are prepared either by ion exchange chromatography
or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-
forming inorganic or organic acid in a suitable solvent or various combinations of solvents.
Similarly, the salts of the acidic compounds are formed by reactions with the appropriate inorganic
or organic base.
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Thus, pharmaceutically acceptable salts of the cationic lipids include non-toxic salts of the
cationic lipids as formed by reacting a basic instant cationic lipids with an inorganic or organic
acid. acid. For For example, example, non-toxic non-toxic salts salts include include those those derived derived from from inorganic inorganic acids acids such such as as hydrochloric, hydrochloric,
hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like, as well as salts prepared from
organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,
ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-
acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,
and trifluoroacetic and trifluoroacetic (TFA). (TFA).
When the cationic lipids are acidic, suitable "pharmaceutically acceptable salts" refers to
salts prepared form pharmaceutically acceptable non-toxic bases including inorganic bases and
organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper,
ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, and zinc. In
one embodiment, the base is selected from ammonium, calcium, magnesium, potassium and
sodium. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of
primary, secondary and tertiary amines, substituted amines including naturally occurring
substituted amines, cyclic amines and basic ion exchange resins, such as arginine, betaine caffeine,
N,N¹-dibenzylethylenediamine,diethylamin, choline, ,N1-dibenzylethylenediamine, diethylamin,2-diethylaminoethanol, 2-diethylaminoethanol, 2-
dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,
glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine,
morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine,
triethylamine, trimethylamine tripropylamine, and tromethamine.
It will also be noted that the cationic lipids may potentially be internal salts or zwitterions,
since under physiological conditions a deprotonated acidic moiety in the compound, such as a
carboxyl group, may be anionic, and this electronic charge might then be balanced off internally
against the cationic charge of a protonated or alkylated basic moiety, such as a quaternary nitrogen
atom.
One or more additional cationic lipids, which carry a net positive charge at about
physiological pH, in addition to those specifically described above, may also be included in the
lipid particles and compositions described herein. Such cationic lipids include, but are not limited
-59- to N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl-N,N-N-triethylammonium N-(2,3-dioleyloxy)propyl-N,N-N-triethylammonium chloride ("DOTMA"); I,N-distearyl-N,N-dimethylammonium N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammoniun N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTAP"); 1,2-Dioleyloxy-3-trimethylaminopropane 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt ("DOTAP.CI");
3B-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholestero 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"),
N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium
trifluoracetate ("DOSPA"), dioctadecylamidoglycyl carboxyspermine ("DOGS"), 1,2-dileoyl-sn-3-phosphoethanolamine ("DOPE"), 1,2-dioleoyl-3-dimethylammonium propane
("DODAP"), N, N-dimethyl-2,3-dioleyloxy)propylamine ("DODMA"), and
IN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide ("DMRIE"). ammonium bromide ("DMRIE").
Additionally, a number of commercial preparations of cationic lipids can be used, such as, e.g.,
LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPA and DOPE, available from GIBCO/BRL).
The Other Lipid Components
The The lipid lipidparticles particlesandand compositions described compositions herein herein described may alsomay include also one or moreone include neutral or more neutral
lipids. Neutral lipids, when present, can be any of a number of lipid species which exist either in
an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, for example,
diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin,
dihydrosphingomyelin, cephalin, and cerebrosides. In one embodiment, the neutral lipid
component is a lipid having two acyl groups (e.g., diacylphosphatidylcholine and
diacylphosphatidylethanolamine). In one embodiment, the neutral lipid contains saturated fatty
acids with carbon chain lengths in the range of C10 C toto C.C20. In another In another embodiment, embodiment, the the neutral neutral
lipid includes mono or diunsaturated fatty acids with carbon chain lengths in the range of C10 C toto
C20. Suitable C. Suitable neutral neutral lipids lipids include, include, but but are are not not limited limited to, to, DSPC, DSPC, DPPC, DPPC, POPC, POPC, DOPE, DOPE, DSPC, DSPC,
and SM.
The lipid particles and compositions described herein may also include one or more lipids
capable of reducing aggregation. Examples of lipids that reduce aggregation of particles during - 60
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formation include polyethylene glycol (PEG)-modified lipids (PEG lipids, such as PEG-DMG and
PEG-DMA), monosialoganglioside Gml, Gm1, and polyamide oligomers ("PAO") such as (described
in U.S. Patent No. 6,320,017, which is incorporated by reference in its entirety). Suitable PEG
lipids include, but are not limited to, PEG-modified phosphatidylethanolamine and phosphatidic
acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) (such as those described in
U.S. Patent No. 5,820,873, incorporated herein by reference), PEG-modified dialkylamines and
PEG-modified 1,2-diacyloxypropan-3-amines, PEG-modified diacylglycerols and and dialkylglycerols, mPEG (mw2000)-diastearoylphosphatidylethanolamine (mw2000)-diastearoylphosphatidylethanolamine.(PEG-DSPE). (PEG-DSPE).
The lipid particles and compositions may include a sterol, such as cholesterol.
Lipid Particles
In a further aspect, the present invent relates to lipid particles that include one or more of
the cationic lipids described herein.
Lipid particles include, but are not limited to, liposomes. As used herein, a liposome is a
structure having lipid-containing membranes enclosing an aqueous interior.
Another embodiment is a nucleic acid-lipid particle (e.g., a SNALP) comprising a cationic
lipid, a non-cationic lipid (such as a neutral lipid), optionally a PEG-lipid conjugate (such as the
lipids for reducing aggregation of lipid particles discussed herein), optionally a sterol (e.g.,
cholesterol), and a nucleic acid. As used herein, the term "SNALP" refers to a stable nucleic acid-
lipid particle. A SNALP represents a particle made from lipids, wherein the nucleic acid (e.g., an
interfering RNA) is encapsulated within the lipids. In certain instances, SNALPs are useful for
systemic applications, as they can exhibit extended circulation lifetimes following intravenous
(i.v.) injection, they can accumulate at distal sites (e.g., sites physically separated from the
administration site), and they can mediate silencing of target gene expression at these distal sites.
The nucleic acid may be complexed with a condensing agent and encapsulated within a SNALP
as set forth in International Publication No. WO 00/03683, the disclosure of which is herein
incorporated by reference in its entirety.
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For example, the lipid particle may include a cationic lipid, a fusion-promoting lipid (e.g.,
DPPC), a neutral lipid, cholesterol, and a PEG-modified lipid. In one embodiment, the lipid
particle includes the above lipid mixture in molar ratios of about 20-70% cationic lipid: 0.1-50%
fusion promoting lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid
(based upon 100% total moles of lipid in the lipid particle).
In another embodiment of the lipid particle, the cationic lipid is present in a mole
percentage of about 20% and about 60%; the neutral lipid is present in a mole percentage of about
5% to about 25%; the sterol is present in a mole percentage of about 25% to about 55%; and the
PEG lipid is PEG-DMA, PEG-DMG, or a combination thereof, and is present in a mole percentage
of about 0.5% to about 15% (based upon 100% total moles of lipid in the lipid particle).
In particular embodiments, the molar lipid ratio, with regard to mol% cationic
lipid/DSPC/Chol/PEG-DMG lipid/DSPC/Chol/PEG-DMG or or PEG-DMA) PEG-DMA) is is approximately approximately 40/10/40/10, 40/10/40/10, 35/15/40/10 35/15/40/10 or or
52/13/30/5. This mixture may be further combined with a fusion-promoting lipid in a molar ratio
of 0.1-50%, 0.1-50%, 0.5-50%, 1-50%, 5%-45%, 10%-40%, or 15%-35%. In other words, when
a 40/10/40/10 mixture of lipid/DSPC/Chol/PEG-DMG or PEG-DMA is combined with a fusion-
promoting peptide in a molar ratio of 50%, the resulting lipid particles can have a total molar ratio
of (mol% cationic lipid/DSPC/Chol/PEG-DMG or PEG-DMA/fusion-promoting peptide)
20/5/20/5/50. In another embodiment, the neutral lipid, DSPC, in these compositions is replaced
with POPC, DPPC, DOPE or SM.
In one embodiment, the lipid particles comprise a cationic lipid, a neutral lipid, a sterol and
a PEG-modified lipid. In one embodiment, the lipid particles include from about 25% to about
75% on a molar basis of cationic lipid, e.g., from about 35 to about 65%, from about 45 to about
65%, about 60%, about 57.5%, about 57.1%, about 50% or about 40% on a molar basis. In one
embodiment, the lipid particles include from about 0% to about 15% on a molar basis of the neutral
lipid, e.g., from about 3 to about 12%, from about 5 to about 10%, about 15%, about 10%, about
7.5%, about 7.1% or about 0% on a molar basis. In one embodiment, the neutral lipid is DPPC. In
one embodiment, the neutral lipid is DSPC.
In one embodiment, the formulation includes from about 5% to about 50% on a molar basis
of the sterol, e.g., about 15 to about 45%, about 20 to about 40%, about 48%, about 40%, about
PCT/US2019/053617
38.5%, about 35%, about 34.4%, about 31.5% or about 31% on a molar basis. In one embodiment,
the sterol is cholesterol.
The lipid particles described herein may further include one or more therapeutic agents. In
a preferred embodiment, the lipid particles include a nucleic acid (e.g., an oligonucleotide), such
as siRNA or miRNA.
In one embodiment, the lipid particles include from about 0.1% to about 20% on a molar
basis of the PEG-modified lipid, e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 10%,
about 5%, about 3.5%, about 1.5%, about 0.5%, or about 0.3% on a molar basis. In one
embodiment, embodiment,the PEG-modified the lipid PEG-modified is PEG-DMG. lipid is -DMG.In In oneone embodiment, the PEG-modified embodiment, lipid lipid the PEG-modified
is PEG-c-DMA. In one embodiment, the lipid particles include 25-75% of cationic lipid, 0.5-15%
of the neutral lipid, 5-50% of the sterol, and 0.5-20% 0.5- 20%of ofthe thePEG-modified PEG-modifiedlipid lipidon ona amolar molarbasis. basis.
In one embodiment, the lipid particles include 35-65% of cationic lipid, 3-12% of the
neutral lipid, 15-45% of the sterol, and 0.5- 10% of the PEG-modified lipid on a molar basis.
In one embodiment, the lipid particles include 45-65% of cationic lipid, 5-10% of the neutral lipid,
25-40% of the sterol, and 0.5-5% 0.5- 5%of ofthe thePEG-modified PEG-modifiedlipid lipidon ona amolar molarbasis. basis.In Inone oneembodiment, embodiment,
the PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da.
In one embodiment, the PEG modified lipid is PEG-distyryl glycerol (PEG-DSG). In one
preferred embodiment, the PEG modified lipid is 1,2-dimyristoyl-sn-glycerol-methoxy
polyethylene glycol (PEG-DMG), such as PEG-DMG with an average polyethylene glycol
molecular weight of 2000.
In one embodiment, the ratio of lipid:siRNA (weight [mg]: weight [mg]
[mg]:weight [mg] is is at at least least about about
0.5:1, at least about 1:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1,
at least about 6:1, at least about 7:1, at least about 11:1 or at least about 33:1. In one embodiment,
the ratio of lipid: siRNA ratio is between about 1:1 to about 35:1, about 3:1 to about 15:1, about
4:1 to about 15:1, or about 5:1 to about 13:1. In one embodiment, the ratio of lipid:siRNA ratio is
between about 3:1 to about 12:1.
One embodiment is a lipid particle comprising a biodegradable cationic lipid, a neutral
lipid, a sterol, and a lipid capable of reducing aggregation (e.g., PEG-modified lipid), where the
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molar ratio of the biodegradable cationic lipid to the sterol ranges from about 1.6:1 to about 2.0:1
and/or the molar ratio of the biodegradable cationic lipid to the neutral lipid ranges from about
5.5:1 to about 5.9:1. The inventors have surprisingly found that lipid particles having certain
higher contents of biodegradable cationic lipid relative to the amount sterol and/or neutral lipid
exhibit enhanced efficacy for the delivery of an active agent (e.g., siRNA). In one embodiment,
the biodegradable cationic lipid comprises a lipid moiety, where the lipid moiety has one or more
-OC(O)-). In one preferred biodegradable groups (such as an ester group (-C(O)O- or -0C(0)-).
embodiment, the lipid capable of reducing aggregation is 1,2-dimyristoyl-sn-glycerol-methoxy
polyethylene glycol (PEG-DMG), such as PEG-DMG with an average polyethylene glycol
molecular weight of 2000.
In a further embodiment, the molar ratio of the biodegradable cationic lipid to the sterol is
from about 1.7 to about 1.9:1, such as about 1.9:1. In another embodiment, the molar ratio of the
biodegradable cationic lipid to the neutral lipid ranges from about 5.5:1 to about 5.8:1, such as
about 5.8:1.
In one embodiment, the lipid particle comprises from about 55 to about 60 mol % of the
biodegradable cationic lipid, such as about 58 mol % (based on 100 mol % of the lipid components
in the lipid particle). The lipid particle may comprise from about 28 to about 33 mol % of the
sterol, such as from about 28 to about 32 mol % (based on 100 mol % of the lipid components in
the lipid particle). In one embodiment, the lipid particle comprises from about 3 to about 12 mol
%, such as from about 5 to about 12 mol %, from about 8 to about 12 mol %, or from about 9 to
about 11 mol %, of the neutral lipid (based on 100 mol % of the lipid components in the lipid
particle). In another embodiment, the lipid particle comprises about 10 mol % of the neutral lipid
(based on 100 mol % of the lipid components in the lipid particle). In yet another embodiment,
the lipid particle comprises from about 0.5 to about 10 mol %, such as from about 0.5 to about 5
mol % or from about 1 to about 3 mol %, of the lipid capable of reducing aggregation (e.g., a PEG-
modified lipid) (based on 100 mol % of the lipid components in the lipid particle).
Another embodiment is a lipid particle comprising a biodegradable cationic lipid, a neutral
lipid, a sterol, and a lipid capable of reducing aggregation (e.g., PEG-modified lipid), where the
lipid particle comprises from about 55 to about 60 mol % of the biodegradable cationic lipid and
WO wo 2020/072324 PCT/US2019/053617 PCT/US2019/053617
from about 33 to about 28 mol % of the sterol (based on 100 mol % of the lipid components in the
lipid particle). In one embodiment, the lipid particle comprises about 58 mol % of the
biodegradable cationic lipid (based on 100 mol % of the lipid components in the lipid particle). In
another embodiment, the lipid particle comprises from about 3 to about 12% of the neutral lipid,
and from about 0.5 to about 10 mol % of the lipid capable of reducing aggregation (based on 100
mol % of the lipid components in the lipid particle). In yet another embodiment, the lipid particle
comprises about 10 mol % of the neutral lipid (based on 100 mol % of the lipid components in the
lipid particle). In yet another embodiment, the lipid particle comprises about 2 mol % of the lipid
capable of reducing aggregation (based on 100 mol % of the lipid components in the lipid particle).
In one embodiment, the lipid particle comprises from about 55 to about 60 mol % of the cationic
lipid, from about 3 to about 12% of the neutral lipid, from about 28 to about 33 mol % of the sterol,
and from about 0.5 to about 10 mol % of the lipid capable of reducing aggregation (based on 100
mol % of the lipid components in the lipid particle). In yet another embodiment, the lipid particle
comprises about 58% of the cationic lipid, about 10% of the neutral lipid, about 30% of the sterol,
and about 2% of the lipid capable of reducing aggregation (based on 100 mol % of the lipid
components in the lipid particle). In yet another embodiment, the lipid particle comprises about
55% of the cationic lipid, about 10% of the neutral lipid, about 33% of the sterol, and about 2% of
the lipid capable of reducing aggregation (based on 100 mol % of the lipid components in the lipid
particle). In one preferred embodiment, the lipid capable of reducing aggregation is 1,2-
dimyristoyl-sn-glycerol-methoxy polyethylene glycol (PEG-DMG), such as PEG-DMG with an
average polyethylene glycol molecular weight of 2000.
In one embodiment, the lipid particles are nanoparticles. In additional embodiments, the
lipid particles have a mean diameter size of from about 50 nm to about 300 nm, such as from about
50 nm to about 250 nm, for example, from about 50 nm to about 200 nm.
In one embodiment, a lipid particle containing a cationic lipid of any of the embodiments
described herein has an in vivo half life (t1/2) (e.g., (t/) (e.g., inin the the liver, liver, spleen spleen oror plasma) plasma) ofof less less than than about about
3 hours, such as less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, less
than about 1 hour, less than about 0.5 hour or less than about 0.25 hours.
WO wo 2020/072324 PCT/US2019/053617
In another embodiment, a lipid particle containing a cationic lipid of any of the
embodiments described herein has an in vivo half life (t1/2) (e.g., (t/) (e.g., inin the the liver, liver, spleen spleen oror plasma) plasma) ofof
less than about 10 % (e.g., less than about 7.5%, less than about 5%, less than about 2.5%) of that
for the same cationic lipid without the biodegrable group or groups.
Additional Components
The lipid particles and compositions described herein can further include one or more
antioxidants. The antioxidant stabilizes the lipid particle and prevents, decreases, and/or inhibits
degradation of the cationic lipid and/or active agent present in the lipid particles. The antioxidant
can be a hydrophilic antioxidant, a lipophilic antioxidant, a metal chelator, a primary antioxidant,
a secondary antioxidant, salts thereof, and mixtures thereof. In certain embodiments, the
antioxidant comprises a metal chelator such as EDTA or salts thereof, alone or in combination
with one, two, three, four, five, six, seven, eight, or more additional antioxidants such as primary
antioxidants, secondary antioxidants, or other metal chelators. In one preferred embodiment, the
antioxidant comprises a metal chelator such as EDTA or salts thereof in a mixture with one or
more primary antioxidants and/or secondary antioxidants. For example, the antioxidant may
comprise a mixture of EDTA or a salt thereof, a primary antioxidant such as a-tocopherol or a salt
thereof, and a secondary antioxidant such as ascorbyl palmitate or a salt thereof. In one
embodiment, the antioxidant comprises at least about 100 mM citrate or a salt thereof. Examples
of antioxidants include, but are not limited to, hydrophilic antioxidants, lipophilic antioxidants,
and mixtures thereof. Non-limiting examples of hydrophilic antioxidants include chelating agents
(e.g., metal chelators) such as ethylenediaminetetraacetic acid (EDTA), citrate, ethylene glycol
tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), (BAPTA),
diethylene triamine pentaacetic acid (DTPA), 2,3-dimercapto-l-propanesulfonic 2,3-dimercapto-I-propanesulfonic acid (DMPS),
dimercaptosuccinic acid (DMSA), cc-lipoic acid, salicylaldehyde isonicotinoyl hydrazone (SIH),
hexyl thioethylamine hydrochloride (HTA), desferrioxamine, salts thereof, and mixtures thereof.
Additional hydrophilic antioxidants include ascorbic acid, cysteine, glutathione, dihydrolipoic
acid, 2- mercaptoethane sulfonic acid, 2-mercaptobenzimidazole sulfonic acid, 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid, sodium metabisulfite, salts thereof, and mixtures thereof.
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Non-limiting Non-limitingexamples of lipophilic examples antioxidants of lipophilic include include antioxidants vitamin Evitamin isomers Esuch as a-,such isomers B-, Y-, and ß-, -, and as -,
S-tocopherols and -tocopherols anda-, -, B-, ß-,Y-, -, and and8-tocotrienols; -tocotrienols; polyphenols such such polyphenols as 2-tert-butyl-4-methyl phenol, phenol, as 2-tert-butyl-4-methyl
2-fert-butyl-5-methyl phenol, and 2-tert-butyl-6-methyl phenol; butylated hydroxyanisole (BHA)
(e.g., 2-teri-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole); 3-tert-butyl-4-hydroxyanisole): butylhydroxytoluene
(BHT); tert-butylhydroquinone (TBHQ); ascorbyl palmitate; rc-propyl gallate; salts thereof; and
mixtures thereof. Suitable antioxidants and formulations containing such antioxidants are
described in International Publication No. WO 2011/066651, which is hereby incorporated by
reference.
In another embodiment, the lipid particles or compositions contain the antioxidant EDTA
(or a salt thereof), the antioxidant citrate (or a salt thereof), or EDTA (or a salt thereof) in
combination with one or more (e.g., a mixture of) primary and/or secondary antioxidants such as
a-tocopherol (or aa salt -tocopherol (or salt thereof) thereof) and/or and/or ascorbyl ascorbyl palmitate palmitate (or (or aa salt salt thereof). thereof).
In one embodiment, the antioxidant is present in an amount sufficient to prevent, inhibit,
or reduce the degradation of the cationic lipid present in the lipid particle. For example, the
antioxidant may be present at a concentration of at least about or about 0.1 mM, 0.5 mM, 1 mM,
10 mM, 100 mM, 500 mM, 1 M, 2 M, or 5M, or from about 0.1 mM to about 1 M, from about 0.1
mM to about 500 mM, from about 0.1 mM to about 250 mM, or from about 0.1 mM to about 100
mM.
The lipid particles and compositions described herein can further include an
apolipoprotein. As used herein, the term "apolipoprotein" or "lipoprotein" refers to apolipoproteins
known to those of skill in the art and variants and fragments thereof and to apolipoprotein agonists,
analogues or fragments thereof described below.
In a preferred embodiment, the active agent is a nucleic acid, such as a siRNA. For
example, the active agent can be a nucleic acid encoded with a product of interest, including but
not limited to, RNA, antisense oligonucleotide, an antagomir, a DNA, a plasmid, a ribosomal RNA
(rRNA), a micro RNA (miRNA) (e.g., a miRNA which is single stranded and 17-25 nucleotides
in length), transfer RNA (tRNA), a small interfering RNA (siRNA), small nuclear RNA (snRNA),
antigens, fragments thereof, proteins, peptides, vaccines and small molecules or mixtures thereof.
WO wo 2020/072324 PCT/US2019/053617 PCT/US2019/053617
In one more preferred embodiment, the nucleic acid is an oligonucleotide (e.g., 15-50 nucleotides
in length (or 15-30 or 20-30 nucleotides in length)). An siRNA can have, for instance, a duplex
region that is 16-30 nucleotides long. In another embodiment, the nucleic acid is an
immunostimulatory oligonucleotide, decoy oligonucleotide, supermir, miRNA mimic, or miRNA
inhibitor. A supermir refers to a single stranded, double stranded or partially double stranded
oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or both or
modifications modifications thereof, thereof, which which has has aa nucleotide nucleotide sequence sequence that that is is substantially substantially identical identical to to an an miRNA miRNA
and that is antisense with respect to its target. miRNA mimics represent a class of molecules that
can be used to imitate the gene silencing ability of one or more miRNAs. Thus, the term
"microRNA mimic" refers to synthetic non-coding RNAs (i.e. the miRNA is not obtained by
purification from a source of the endogenous miRNA) that are capable of entering the RNAi
pathway and regulating gene expression.
The nucleic acid that is present in a lipid-nucleic acid particle can be in any form. The
nucleic acid can, for example, be single-stranded DNA or RNA, or double-stranded DNA or RNA,
or DNA-RNA hybrids including their chemically modified analogs. Non-limiting examples of
double-stranded RNA include siRNA. Single-stranded nucleic acids include, e.g., antisense
oligonucleotides, ribozymes, microRNA, and triplex-forming oligonucleotides. The lipid particles
can also deliver nucleic acids which are conjugated to one or more ligands.
Pharmaceutical Compositions
The lipid particles, particularly when associated with a therapeutic agent, may be
formulated as a pharmaceutical composition, e.g., which further comprises a pharmaceutically
acceptable diluent, excipient, or carrier, such as physiological saline or phosphate buffer.
The resulting pharmaceutical preparations may be sterilized by conventional, well known
sterilization techniques. The aqueous solutions can then be packaged for use or filtered under
aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile
aqueous solution prior to administration. The compositions may contain pharmaceutically
acceptable auxiliary substances as required to approximate physiological conditions, such as pH
PCT/US2019/053617
adjusting and buffering agents, and tonicity adjusting agents, for example, sodium acetate, sodium
lactate, sodium chloride, potassium chloride, and calcium chloride. Additionally, the lipidic
suspension may include lipid-protective agents which protect lipids against free-radical and
a-tocopherol lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as -tocopherol
and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.
The concentration of lipid particle or lipid-nucleic acid particle in the pharmaceutical
formulations can vary, for example, from less than about 0.01%, to at or at least about 0.05-5% to
as much as 10 to 30% by weight.
Methods of Manufacture
Methods of making cationic lipids, lipid particles containing them, and pharmaceutical
compositions containing the cationic lipids and/or lipid particles are described in, for example,
International Publication Nos. WO 2010/054406, WO 2010/054401, WO 2010/054405, WO
2010/054384, WO 2010/042877, WO 2010/129709, WO 2009/086558, and WO 2008/042973,
and U.S. Patent Publication Nos. 2004/0142025, 2006/0051405 and 2007/0042031, each of which
is incorporated by reference in its entirety.
For example, in one embodiment, a solution of one or more lipids (including a cationic
lipid of any of the embodiments described herein) in an organic solution (e.g., ethanol) is prepared.
Similarly, a solution of one or more active (therapeutic) agents (such as, for example an siRNA
molecule or a 1:1 molar mixture of two siRNA molecules) in an aqueous buffered (e.g., citrate
buffer) solution is prepared. The two solutions are mixed and diluted to form a colloidal suspension
of siRNA lipid particles. In one embodiment, the siRNA lipid particles have an average particle
size of about 80-90 nm. In further embodiments, the dispersion may be filtered through 0.45/2
micron filters, concentrated and diafiltered by tangential flow filtration.
WO wo 2020/072324 PCT/US2019/053617 PCT/US2019/053617
Definitions
As used herein, the term "cationic lipid" inlcudes those lipids having one or two fatty acid
or fatty aliphatic chains and an amino acid containing head group that may be protonated to form
a cationic lipid at physiological pH. In some embodiments, a cationic lipid is referred to as an
"amino acid conjugate cationic lipid."
A subject or patient in whom administration of the complex is an effective therapeutic
regimen for a disease or disorder is preferably a human, but can be any animal, including a
laboratory animal in the context of a clinical trial or screening or activity experiment. Thus, as
can be readily appreciated by one of ordinary skill in the art, the methods, compounds and
compositions of the present invention are particularly suited to administration to any animal,
particularly a mammal, and including, but by no means limited to, humans, domestic animals, such
as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine,
and porcine subjects, wild animals (whether in the wild or in a zoological garden), research
animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, and cats, avian species, such as
chickens, turkeys, and songbirds, i.e., for veterinary medical use.
Many of the chemical groups recited in the generic formulas above are written in a
particular order (for example, -OC(O)-). It is intended that the chemical group is to be incorporated
into the generic formula in the order presented unless indicated otherwise. For example, a generic
formula of the form -(R);-(M )k-(R)m- -(R);-(M¹)-(R)m where where M¹M1 isis -C(O)O- -C(0)0- and and k k isis 1 1 refers refers toto -(R)i-C(0)0- -(R);-C(O)O-
(R)m- unless specified (R)- unless specified otherwise. otherwise. It It is is to to be be understood understood that that when when aa chemical chemical group group is is written written in in aa
particular order, the reverse order is also contemplated unless otherwise specified. For example,
in a generic formula -(R);-(M 1)-(R)m- -(R);-(M¹)-(R)m where where M¹Mis ¹ is defined defined as as -C(O)NH- -C(O)NH- (i.e., (i.e., -(R)i-C(O)-NH- -(R);-C(O)-NH-
(R)m-), the compound where M M¹¹ is is -NHC(O)- -NHC(O)- (i.e., (i.e., -(R);-NHC(O)-(R)r) -(R);-NHC(O)-(R)m) is is also also contemplated contemplated
unless otherwise specified.
The term "biodegradable cationic lipid" refers to a cationic lipid having one or more
biodegradable groups located in the mid- or distal section of a lipidic moiety (e.g., a hydrophobic
chain) of the cationic lipid. The incorporation of the biodegradable group(s) into the cationic lipid
results in faster metabolism and removal of the cationic lipid from the body following delivery of
the active pharmaceutical ingredient to a target area.
- 70
As used herein, the term "biodegradable group" refers to a group that include one or more
bonds that may undergo bond breaking reactions in a biological environment, e.g., in an organism,
organ, tissue, cell, or organelle. For example, the biodegradable group may be metabolizable by
the body of a mammal, such as a human (e.g., by hydrolysis). Some groups that contain a
biodegradable bond include, for example, but are not limited to esters, dithiols, and oximes. Non-
limiting examples of biodegradable groups are -OC(O)-, -C(O)O-, -C(0)0-, -SC(O)-, -C(O)S-, -OC(S)-, - -
C(S)O-, C(S)O-, -S-S-, -S-S-,-C(R5)=N-, -C(R)=N-,-N=C(R5)-, -N=C(R)-,-C(R5)=N-O-, -C(R)=N-O-,-O-N=C(R5)-, -C(O)(NR³), -O-N=C(R)-, -N(R5)(())- -C(O)(NR)-, -N(R)C(O)-, - -
C(S)(NR5)-,-N(R5)C(O)-,-N(R5)C(O)N(R5)-,-OC(O)O-,-OSi(R5)2O-,-C(O)(CR3R4)C(O)O-, C(S)(NR)-, -N(R)C(O)-, -N(R°)C(O)N(R³)-, -0C(0)0-, -OSi(R)O-, -C(O)(CR³R)C(O)O-, or or
-OC(O)(CR³R)C(O)-.
As used herein, an "aliphatic" group is a non-aromatic group in which carbon atoms are
linked into chains and is either saturated or unsaturated.
The terms "alkyl" and "alkylene" refer to a straight or branched chain saturated
hydrocarbon moiety. In one embodiment, the alkyl group is a straight chain saturated hydrocarbon.
Unless otherwise specified, the "alkyl" or "alkylene" group contains from 1 to 24 carbon atoms.
Representative saturated straight chain alkyl groups include methyl, ethyl, in-propyl, in-butyl, n-propyl, n-butyl,
in-pentyl, andn-hexyl. n-pentyl, and n-hexyl.Representative Representativesaturated saturatedbranched branchedalkyl alkylgroups groupsinclude includeisopropyl, isopropyl,
sec-butyl, isobutyl, tert-butyl, and isopentyl.
The term "alkenyl" refers to a straight or branched chain hydrocarbon moiety having one
or more carbon-carbon double bonds. In one embodiment, the alkenyl group contains 1, 2, or 3
double bonds and is otherwise saturated. Unless otherwise specified, the "alkenyl" group contains
from 2 to 24 carbon atoms. Alkenyl groups include both cis and trans isomers. Representative
straight chain and branched alkenyl groups include ethylenyl, propylenyl, 1-butenyl, 2-butenyl,
isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, and
2,3-dimethyl-2-butenyl. 2,3-dimethyl-2-butenyl.
The term "alkynyl" refers to a straight or branched chain hydrocarbon moiety having one
or more carbon-carbon triple bonds. Unless otherwise specified, the "alkynyl" group contains
from 2 to 24 carbon atoms. Representative straight chain and branched alkynyl groups include
acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl.
PCT/US2019/053617
Unless otherwise specified, the terms "branched alkyl", "branched alkenyl", and "branched
alkynyl" refer to an alkyl, alkenyl, or alkynyl group in which one carbon atom in the group (1) is
bound to at least three other carbon atoms and (2) is not a ring atom of a cyclic group. For example,
a spirocyclic group in an alkyl, alkenyl, or alkynyl group is not considered a point of branching.
Unless otherwise specified, the term "acyl" refers to a carbonyl group substituted with
hydrogen, alkyl, partially saturated or fully saturated cycloalkyl, partially saturated or fully
saturated heterocycle, aryl, or heteroaryl. For example, acyl groups include groups such as
(C1-C2o)alkanoyl (e.g., (C-C)alkanoyl (e.g., formyl, formyl, acetyl, acetyl, propionyl, propionyl, butyryl, butyryl, valeryl, valeryl, caproyl, caproyl, andand t-butylacetyl), t-butylacetyl),
(C3-C20)cycloalkylcarbonyl (e.g., (C-C)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl, cyclopropylcarbonyl, cyclobutylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclopentylcarbonyl,
cyclohexylcarbonyl), heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl, and
pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, and
tetrahydrofuranylcarbonyl), aroyl (e.g., benzoyl) and heteroaroyl (e.g., thiophenyl-2-carbonyl,
thiophenyl-3-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl, furanyl-3-carbonyl, IH-pyrroyl-2-carbonyl, furanyl-2-carbonyl, furanyl-3-carbonyl, IH-pyrroyl-2-carbonyl,
1H-pyrroyl-3-carbonyl, and 1H-pyrroyl-3-carbonyl, and benzo[b]thiophenyl-2-carbonyl). benzo[b]thiophenyl-2-carbonyl).
The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring
system. Unless otherwise specified, the "aryl" group contains from 6 to 14 carbon atoms.
Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, anthracenyl, and
pyrenyl.
The terms "cycloalkyl" and "cycloalkylene" refer to a saturated monocyclic or bicyclic
hydrocarbon moiety such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Unless
otherwise specified, the "cycloalkyl" or "cycloalkylene" group contains from 3 to 10 carbon atoms.
The The term term"cycloalkylalkyl" "cycloalkylalkyl"refers to a to refers cycloalkyl group bound a cycloalkyl groupto bound an alkyl to group, where an alkyl the group, where the
alkyl group is bound to the rest of the molecule.
The term "heterocycle" (or "heterocyclyl") refers to a non-aromatic 5- to 8-membered
monocyclic, or 7- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system which is
either saturated or unsaturated, and which contains from 1 to 3 heteroatoms if monocyclic, 1-6
heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, independently selected from nitrogen,
oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized,
-72- and the nitrogen heteroatom may be optionally quaternized. For instance, the heterocycle may be a cycloalkoxy group. The heterocycle may be attached to the rest of the molecule via any heteroatom or carbon atom in the heterocycle. Heterocycles include, but are not limited to, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxiranyl, oxetanyl, oxetanyl, tetrahydrofuranyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, and tetrahydrothiopyranyl.
The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 7-12 membered
bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6
heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, where the heteroatoms are selected from
O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic,
or tricyclic, respectively). The heteroaryl groups herein described may also contain fused rings
that share a common carbon-carbon bond.
The term "substituted", unless otherwise indicated, refers to the replacement of one or more
hydrogen radicals in a given structure with the radical of a specified substituent including, but not
limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, oxo, thioxy, arylthio,
alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy,
aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl,
aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino,
alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl,
alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid,
sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and an aliphatic group. It is understood
that the substituent may be further substituted. Exemplary substituents include amino, alkylamino,
dialkylamino, and cyclic amino compounds.
The term "halogen" or "halo" refers to fluoro, chloro, bromo and iodo.
The following abbreviations may be used in this application:
DSPC: distearoylphosphatidylcholine; DPPC: 1,2-Dipalmitoyl-sn-glycero-3-
phosphocholine; POPC: 1- palmitoyl-2-oleoyl-sn-phosphatidylcholine DOPE: 1,2-dileoyl-sn-3- phosphoethanolamine; PEG-DMG generally refers to 1,2-dimyristoyl-sn-glycerol-methoxy polyethylene glycol (e.g., PEG 2000); TBDPSCI: tert-Butylchlorodiphenylsilane; DMAP: dimethylaminopyridine; HMPA: hexamethylphosphoramide; EDC: 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide; DIPEA: diisopropylethylamine; DCM: dichloromethane;
TEA: triethylamine; TBAF: tetrabutylammonium fluoride
Methods to prepare various organic groups and protective groups are known in the art and
their use and modification is generally within the ability of one of skill in the art (see, for example,
Green, T.W. et. al., Protective Groups in Organic Synthesis (1999); Stanley R. Sandler and Wolf
Karo, Organic Functional Group Preparations (1989); Greg T. Hermanson, Bioconjugate
Techniques (1996); and Leroy G. Wade, Compendium Of Organic Synthetic Methods (1980)).
Briefly, protecting groups are any group that reduces or eliminates unwanted reactivity of a
functional group. A protecting group can be added to a functional group to mask its reactivity
during certain reactions and then removed to reveal the original functional group. In some
embodiments an "alcohol protecting group" is used. An "alcohol protecting group" is any group
which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting
groups can be added and removed using techniques well known in the art.
The compounds may be prepared by at least one of the techniques described herein or
known organic synthesis techniques.
Examples
Example 1: Synthesis of Ether Linked Lipids
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2 O Br- Br Br RuCl3 RuCl OH I' A Br Br- Br o NaH K2CO3, THF O NalO4 KCO, THF HO NalO OH an.tol, an.tol, 80oC, 80oC, o/n o/n 33 DCM/ACN/H2O 1 10C-rt-o/n 44
O O o CI CI OH OH OH HO or or Oxayl Chloride O O N N O CI CI K2CO3 DCM OH DCM, 0C-rt, o/n 6 KCO DCM 5
O ALNY-652 ALNY-659 o O o O N N o O
ALNY-659 O ALNY-651
10-(4-bromobutoxy)nonadeca-1,18-diene 3 3 10-(4-bromobutoxy)nonadeca-1,18-diene
NaH Br Br Br O HO Ho C19H36 O C4H8Br2 C23H43BrO CHO Mol. Wt: 280.50 CHBr Mol. Wt: 215.92 Mol. CHBrO Mol. Wt: Wt: 415.50 415.50
1 2 3
To a solution of nonadeca-1,18-dien-10-ol (30 mmol, 8.145 g) in toluene, added NaH (90 mmol,
60%, 3.6g). 3.6 g).The Thereaction reactionmixture mixturewas wasallowed allowedto tostir stirat at80 80°C °Cfor forovernight. overnight.Cooled Cooledto toroom roomtemperature, temperature,
to the reaction was added neat 1,4-dibromobutane (300 mmol, 64.78 The g). reaction mixture The reaction was then mixture was then
heated at 90 °C for 6 hours. TLC showed the completion of reaction. Cooled to room temperature, the
reaction mixture was quenched with ice, extracted with EtOAc, washed with brine. The organic layer
was separated and dried over sodium sulfate. The organic layer was filtered and evaporated under reduced
pressure. The residue was purified by ISCO (SiO2: 0-10% EtOAc / hexane) to provide the product as a
colorless oil (12.46g). 1H ¹H NMR (400 MHz, Chloroform-d) 8 5.81 5.81 (m, (m, JJ == 16.9, 16.9, 10.2, 10.2, 6.7 6.7 Hz, Hz, 2H), 2H), 5.04 5.04 --
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4.88 (m, 4H), 3.65 - 3.31 (m, 5H), 3.18 (p, J = 5.8 Hz, 1H), 2.09 - 1.98 (m, 5H), 2.02 - 1.90 (m, 3H),
1.75 - 1.63 (m, 2H), 1.51 - 1.19 (m, 21H).
9-(4-bromobutoxy)heptadecanedioic: acid 9-(4-bromobutoxy)heptadecanedioic 4 acid 4
OH O RuCI3 RuCl3 OH Br Br O NalO4 NalO O O O C23H43BrO C21H3gBrO5 CHBrO Mol. Wt: 415.50 CHBrO Mol. Wt: 451.44
3 3 4
To a 1L RBF, added 10-(4-bromobutoxy)nonadeca-1,18-diene (30 mmol, 12.46 g) in 400 mL
(1:1 volume) anhydrous DCM and Acetonitrile, RuC13 RuCl3 (1.5 mmol, 311 mg) was added and cooled in an
ice bath, a solution of NaIO4 (300 mmol, 64 g) in water was added slowly. The reaction mixture was
allowed to stir at room temperature overnight. TLC showed completion of reaction. The reaction mixture
was diluted with DCM and water. A few drops of 3% sodium sulfite were added for de-colorization. The
organic layer was separated and dried over sodium sulfate and evaporated under vacuum. The residue
was purified by ISCO (SiO2: 0-10% MeOH/DCM (+0.1% AcOH)) to provide product as a clolorless oil
(7.9 g). MS: M+2=453.1. 1H ¹H NMR (400 MHz, Chloroform-d) 8 3.44 3.44 (m, (m, JJ == 12.3, 12.3, 6.2 6.2 Hz, Hz, 4H), 4H), 3.18 3.18 (p, (p,
J = 5.8 Hz, 1H), 3.09 (q, J = 7.3 Hz, 3H), 2.32 (t, J = 7.4 Hz, 5H), 2.07 (s, 3H), 2.00 - 1.88 (m, 2H), 1.73
- 1.54 (m, 7H), 1.47-1.39 - (m, 4H), 1.47-1.30(m, 10H). 1.47 - 1.39
9-(4-(dimethylamino)butoxy)heptadecanedioic: acid acid 9-(4-(dimethylamino)butoxy)heptadecanedioic 5 5
Br O O OH IH
K2CO3, THF N O OH O O O C21H3gBrO5 C23H45NO5 CHBrO Mol. Wt: 451.44 Mol.CHNO Mol. Wt.: Wt.: 415.62 415.62
5 5 4
To a 150 mL pressure bottle, added 9-(4-bromobutoxy)heptadecanedioic acid (10 mmol, 4.51 g)
in anhydrous THF (80 mL), followed by potassium carbonate (30 mmol, 4.14 g) and 2M solution of
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dimethylamine (50 mmol, 25 mL) in anhydrous THF. The reaction mixture was sealed and allowed to
stir at 65°C overnight. TLC showed completion of reaction. The reaction mixture was acidified by adding
1N HCI to PH~4, the solution was extracted with DCM and water. The organic layer was separated and
dried over sodium sulfate. The organic solution was filtered and evaporated under vacuum. The residue
was co-evaporated with 3x20 mL toluene to provide product as a light- brown oil (3.64 g).
9-(4-(dimethylamino)butoxy)heptadecanedioic 9-(4-(dimethylamino)butoxy)heptadecanedioic dichloride dichloride 66
CI OH O O Oxayl Chlroide CI OH N N O O DMF, DCM O O
C23H43Cl2NO3 C23H45NO5 CHClNO CHNO Mol. Wt.: 415.62 Mol. Wt.: 452.50
6 5
To a solution of 9-(4-(dimethylamino)butoxy)heptadecanedioic acid (8.75 mmol, 3.64 g)
in anhydrous DCM(50 mL), was added 5 drops of anhydrous DMF at 0 °C, added oxayl chloride
(52.5 mmol, 4.58 mL) dropwise. The reaction mixture was allowed to stir at room temperature
overnight. Reaction was completed by checking MS when prepared sample in MeOH, the mass
spectrum showed methyl ester mass. The reaction mixture was evaporated under vacuum and
provided product as a red oil which was used as such in the next step. MS after treated wth MeOH
( Methylester)MS: (Methylester) MS:M+1 M+1=444.3. =444.3.
bis(3-pentyloctyl) 9-(4-(dimethylamino)butoxy)heptadecanedioate (ALNY-651) -(4-(dimethylamino)butoxy)heptadecanedioate (ALNY-651)
CI CI HO Ho O O CI O N N O O O K2CO3, DCM O O
C23H43Cl2NO3 C49H97NO5 CHClNO Mol. Wt.: 452.50 CHNO Mol. Wt: 780.32
6 6 651
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To a solution of 9-(4-(dimethylamino)butoxy)heptadecanedioyl dichloride (3.29 mmol,
1.49 g) in DCM (30mL) was added potassium carbonate (16.45 mmol, 2.27 g) followed by 3-
pentyloctan-1-ol (13.16 pentyloctan-1-o (13.16mmol, 2.64 mmol, g). g). 2.64 The The reaction mixture reaction was allowed mixture to stir to was allowed at room stir at room
temperature overnight. TLC showed completion of reaction. The reaction was quenched with
water and extracted with DCM. The organic layer was separated and dried over sodium sulfate
and evaporated under vacuum. The residue was purified by ISCO (SiO2: 30-100% EtOAc/hexane
¹H NMR (500 MHz, with 3% TEA) to provide a colorless oil (0.538 g). MS: M+1=780.7. 1H
Chloroform-d) 8 4.07 4.07 (t, (t, JJ == 7.1 7.1 Hz, Hz, 6H), 6H), 3.40 3.40 (s, (s, 2H), 2H), 2.35 2.35 -- 2.23 2.23 (m, (m, 10H), 10H), 2.21 2.21 (s, (s, 9H), 9H), 1.78 1.78 (p, (p,
J = 7.5 Hz, 2H), 1.57 (tt, J = 13.8, 6.9 Hz, 17H), 1.46 - 1.36 (m, 8H), 1.29 (t, J = 6.9 Hz, 26H),
0.88 (t, J = 7.1 Hz, 17H).
bis(3-pentyloctyl)9-(4-(isopropyl(methyl)amino)butoxy)heptadecanedioate bis(3-pentyloctyl) 9-(4-(isopropyl(methyl)amino)butoxy)heptadecanedioate(ALNY- (ALNY-
659) 659)
N O O C51H101NO5 CHNO Mol. Wt: 808.37
659 659 The lipid 659 was synthesized using a similar procedure. MS: M+1=809.7. 1H ¹H NMR (500
MHz, MHz, Chloroform-d) Chloroform-d)8 4.08 (t,(t, 4.08 J = J7.1 Hz, 6H), = 7.1 3.40 (t, Hz, 6H), J =(t, 3.40 5.9JHz, 2H),Hz, = 5.9 3.182H), (s, 1H), 3.18 2.39 (s, -1H), 2.24 2.39 - 2.24
(m, 10H), 2.19 (s, 4H), 2.18(m, 1H), 1.65 - 1.46 (m, 19H), 1.40 (dq, J = 12.6, 7.5 Hz, 9H), 1.30
(d, J = 7.6 Hz, 25H), 0.98 (t, J = 5.9 Hz, 8H), 0.88 (t, J = 7.0 Hz, 17H).
bis(2-isopropyl-5-methylhexyl) 9-(4-(dimethylamino)butoxy)heptadecanedioate bis(2-isopropyl-5-methylhexyl) -(4-(dimethylamino)butoxy)heptadecanedioate
(ALNY-652)
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C43H85NO5 CHNO Mol. Wt: 696.16
652 MS: M+1=697.6. 1H ¹H NMR (400 MHz, Chloroform-d) 84.09 4.09--3.88 3.88(m, (m,4H), 4H),3.40 3.40(s, (s,
2H), 3.18(t, 1H) 2.33 - 2.23 (m, 6H), 2.20 (s, 6H) 1.76 (pd, J = 6.9, 4.7 Hz, 4H), 1.66 - 1.09 (m,
40H), 0.88 (td, J = 6.5, 5.9, 3.0 Hz, 22H). ¹³C NMR (126 MHz, Chloroform-d) 174.27 79.68 40H), NMR (126 MHz, Chloroform-d) 8 174.27 , 79.68 , 68.87 , 65.54 65.54 , 59.93 59.93 45.69 45.69, 43.55 43.55, 36.98 36.98 34.70 34.67, 34.70, 34.67 34.23 29.93 , 29.59 29.52, 29.59, 29.52 29.39, 29.39
28.64 28.64,, 28.51 28.51 ,, 28.37 28.37, 26.06 26.06 25.63 25.6325.23 25.2325.19 , 24.75 25.19 24.7522.92 22.92, 22.68 22.68 ,,19.64. 19.64.
bis(2-isopropyl-5-methylhexyl)S 9-(4-(isopropyl(methyl)amino)butoxy) bis(2-isopropyl-5-methylhexyl) -(4-(isopropyl(methyl)amino)butoxy)
heptadecanedioate (ALNY-657)
C45H89NO5 CHNO Mol. wt: 724.21
657 MS: M+1=725.0. 1H ¹H NMR (400 MHz, Chloroform-d) 8 4.07 54.07-3.95 - 3.95 - (m, (m, 4H), 4H), 3.40 3.40 (s, (s,
2H), 3.18 (s, 1H), 2.36 (s, 2H), 2.29 (t, J = 7.5 Hz, 4H), 2.19 (s, 3H), 1.76 (qd, J = 6.9, 4.7 Hz,
3H), 1.56 - 1.10 (m, 42H), 0.98 (dd, J = 6.6, 4.6 Hz, 6H), 0.88 (td, J = 6.5, 5.9, 3.0 Hz, 22H).
O O-P O O o O Pd/C O
H2 balloon NaHMDS/THE NaHMDS/THF |I II II III III
- 79 wo 2020/072324 WO PCT/US2019/053617 PCT/US2019/053617
Ethyl 3-pentyloct-2-enoate II
rojajan O O O NaHMDS/THF
C11H22' C15H28O2 CHO Mol. Wt: 170.30 CHO Mol. Wt: 240.39
I Il II
To a solution of triethyl phosphonacetate(100 phosphonacetate( 100mmol, mmol,22.42 22.42g) g)in inanhydrous anhydrousTHF(50 THF(50mL) mL)
and at -10°C, added - -10°C, 1N 1N added NaHMDS (IN NaHMDS in in (IN THF, 100 THF, mL) 100 via mL) addition via funnel addition dropwise. funnel After dropwise. the After the
addition, the reaction mixture was stirred at -10 °C for 1hr then 0 °C for 1hr. To this added 6-
Undecanone (50 mmol, 8.52 g), warmed to room temperature and stirred at 45°C overnight. The
reaction mixture was quenched with water, extracted with diethylether (2X100 mL), the combined
ether was washed with brine. The organic layer was dried over sodium sulfate and filtered. The
organic solution was evaporated. The residue was purified by ISCO (SiO2: 100% Hexane) to
provide product as a clolorless oil (11.7 g). ¹H 1H NMR (400 MHz, Chloroform-d) 85.61 5.61(s, (s,1H), 1H),
4.13 (q, J = 7.1 Hz, 2H), 2.66 - 2.49 (m, 2H), 2.12 (td, J = 7.6, 1.2 Hz, 2H), 1.45 (ddt, J = 10.1,
7.3, 7.3, 4.5 4.5Hz, Hz,4H), 1.38 4H), - 1.21 1.38 (m, 10H), - 1.21 0.94 -0.94 (m, 10H), 0.84 -(m, 7H).(m, 7H). 0.84
Ethyl 3-pentyloctanoate III
O Pd/C Pd/C
H2 balloon
C15H28O2 C15H30O2 CHO Mol. Wt: 240.39 CHO Mol. Wt: 242.40
To a 200mL RBF, a solution of ethyl 3-pentyloct-2-enoate (48.6 mmol, 11.7 g) in ethyl
acetate (100 mL) was added. The solution was purged the solution with argon 3 times, added Pd/C
(5% wt), purged and backfilled with argon followed by hydrogen via hydrogen balloons. The
- 80 reaction mixture was stirred with H2 balloon overnight. Filtered through celite, washed with thoroughly with ethyl acetate, the combined organic solution was evaporated under reduced
1H NMR (400 MHz, Chloroform-d) 84.12 pressure to provide product as a colorless oil (11 g). ¹H 4.12
(q, J = 7.1 Hz, 2H), 2.21 (d, J = 6.9 Hz, 2H), 1.84 (p, J = 5.8 Hz, 1H), 1.27 (dddd, J = 14.1, 11.3,
7.1, 4.8 Hz, 19H), 0.87 (t, J = 6.9 Hz, 6H).
3-Pentyloctan-1-ol IV 3-Pentyloctan-1-0l
O OH LAH THF C13H28' O C15H30O2
CHO Mol. Wt: 242.40 CHO Mol. Wt: 200.37
To To aa 250mL 250mLRBF, RBF,added a solution added of ethyl a solution 3-pentyloctanoate of ethyl (45.4 mmol, 3-pentyloctanoate 11 mmol, (45.4 g) in 11 g) in
anhydrous THF. Cooled in an ice bath and under argon, added LAH (2N THF, 91 mmol, 45.4 mL)
slowly. Warmed to room temperature and allowed to stir at room temperature for 30min then 70°C
for overnight. The reaction mixture was cooled to room temperature. At 0 °C, a solution of sat.
sodium potassium tartrate tetrahydrate was added dropwise until no bubbling. The mixture was
diluted with diethylether, filtered through celite, the combined ether was dried over sodium sulfate
and filtered. The organic solution was evaporated under reduced pressure. The residue was purified
1H NMR (400 MHz, by ISCO(SiO2: 0-10% EtOAc/Hexane) to provide product 8.26 g. ¹H Chloroform-d) 83.61 3.61(t, (t,JJ==7.1 7.1Hz, Hz,2H), 2H),2.08 2.08(s, (s,1H), 1H),1.49 1.49(q, (q,JJ==6.9 6.9Hz, Hz,2H), 2H),1.38 1.38(p, (p,JJ==4.9, 4.9,
4.0 Hz, 1H), 1.33-1.16 - (m, 16H), 0.86 (t, J = 6.9 Hz, 6H). 1.33 - 1.16
Example 2: Synthesis of Ester Containing Lipids
WO wo 2020/072324 PCT/US2019/053617
1) Swern Oxidation (COCI)2, DMSO
OH 2) 2) Wittig Wittig
Ethoxycarbonylmethylene Triphenylphosphorate
1) 1) LAH LAH MgBr 2) MsCl, TEA N
CuBr, LiCI, TMSCI 3) NMe2, THF
0 O N THF
HCI/Actone N N- N rt, rt, 24hr 24hr
HO Oxone DMF DMF RCOOH
ROCO Oxayl Chloride
Ethyl (2E,13Z,16Z)-docosa-2,13,16-trienoate
Chemical Chemical Formula: C24H42O2 Formula: CHO Molecular Molecular Weight: Weight: 362.60 362.60
To a 1L RBF, added 750mL DCM and with stirring, added Oxayl chloride (239.1 mmol,
20.5 mL). Cooled to -78°C, DMSO (296.48 mmol, 23.16 g) was added dropwise followed by a
solution of (11Z,14Z)-icosa-11,14-dien-1-ol (119.55 mmol, 35.21 g) in DCM (50 mL)slowly in a
period of 5min. After stirring at -78°C for 30min, triethylamine (717.3 mmol, 100 mL) was added.
PCT/US2019/053617
The reaction mixture was warmed to room temperature and stirred for additional 50min. To the
reaction was added ethoxycarbonylmethyl triphenyl phosphonate (135.47 mmol, 51 g) and allowed
to stir at room temperature overnight. TLC showed the completion of reaction. The reaction
mixture was quenched with 1N HCI, extracted with DCM, and washed with brine. The organic
layer was separated and dried over sodium sulfate and evaporated under vacuum. The residue was
purified by ISCO(SiO2: 0-30% EtOAc/Hexane) to provide product as a colorless oil ( 26.75 26.75 g). g).
MS: M+1=363.3. 1H ¹H NMR (400 MHz, Chloroform-d) 6.96 6.96(dt, (dt,JJ==15.7, 15.7,7.0 7.0Hz, Hz,1H), 1H),5.81 5.81(dt, (dt,JJ
= 15.7, 1.6 Hz, 1H), 5.45 - 5.27 (m, 5H), 4.18 (q, J = 7.1 Hz, 2H), 2.77 (t, J = 6.5 Hz, 2H), 2.19
(qd, J = 7.2, 1.6 Hz, 2H), 2.04 (dd, J = 7.6, 6.1 Hz, 5H), 1.44 (q, J = 7.2 Hz, 2H), 1.41 - 1.28 (m,
22H). 22H).
Ethyl (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dienoate
O Chemical ChemicalFormula: Formula:C35H64O4 CHO Molecular Weight: 548.89
To a pre-dried 250mL RBF, added CuBr (0.76 mmol, 109 mg) and LiCl (1.52 mmol, 64.4
mg) in THF (10 mL) and stirred at room temperature for 10 min, cooled in an ice bath, added ethyl
(2E,13Z,16Z)-docosa-2,13,16-trienoate (7.6 mmol, 2.8 g) in DCM (40 mL) followed by TMSCI
(8.36 mmol, 908 mg). The reaction mixture was stirred at 0 °C for 15min before added (8-(1,3-
dioxolan-2-yl)octyl)magnesium bromide (11.4 mmol, 3.3 g) in THF(16 mL). The reaction mixture
was allowed to stir at 0 °C for 1.5hr, and then stirred at room temperature overnight. TLC showed
completion of reaction, the reaction was quenched with sat. NH4Cl, NH4CI, diluted with diethylether (200
mL), extracted and separated, washed with brine. The organic layer was dried sodium sulfate and
evaporated under vacuum. The residue was purified by ISCO (SiO2: 0-20% EtOAc and hexane)
to provide product as a colorless oil (2.16 g). 1H ¹H NMR (500 MHz, Chloroform-d) 8 5.35 5.35 (dtd, (dtd, JJ ==
18.0, 10.6, 5.4 Hz, 4H), 4.84 (t, J = 4.8 Hz, 2H), 4.12 (q, J = 7.2 Hz, 2H), 3.90 (d, J = 59.2 Hz,
6H), 3.56 (t, J = 6.8 Hz, 1H), 2.77 (t, J = 6.9 Hz, 2H), 2.21 (d, J = 6.9 Hz, 2H), 2.05 (q, J = 7.2 Hz,
- 83
4H), 1.83 (t, J = 7.3 Hz, 1H), 1.65 (dt, J = 8.9, 5.1 Hz, 3H), 1.51 (t, J : = 6.8 Hz, 1H), 1.46 - 1.26
(m, 33H), 0.89 (t, J = 6.6 Hz, 3H).
(13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dien-1-ol (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dien-1-ol
O Chemical Chemical Formula: C33H62O3 Formula: CHO Molecular Weight: 506.86
At 0 °C, to a solution of ethyl (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16- 13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-
dienoate (10.87 mmol, 5.97 g) in anhydrous THF(60mL) was added lithium aluminum hydride
(1N in THF, 21.74 mmol, 22 mL) slowly. The reaction mixture was allowed to stir at 0°C for
15min then at room temperature for 4hrs. Cooled in an ice bath, sat. sodium potassium tartrate
tetrahydrate solution was added dropwise until no bubbling. The reaction mixture was diluted with
EtOAc and extracted. The organic layer was separated and dried over sodium sulfate and
concentrated to a colorless oil. The residue was co-evaporated with toluene to remove any trace of
water. Yield product 3.61 g.
(13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dien-1-ylmethanesulfonate (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dien-1-yl methanesulfonate
OMs
O Chemical ChemicalFormula: Formula:C34H64O5S C3HOS Molecular Weight: 584.94
A solution of (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dien-1-o1 (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dien-1-ol (7.132
mmol, 3.61 g) in anhydrous DCM (50 mL) at 0 °C was added trimethylamine (21.39 6mmol, 2.99
mL) followed by MsCl (14.264 mmol, 1.1 mL). The reaction mixture was allowed to stir at room
temperature for 5hrs. TLC showed the completion of reaction. Quenched reaction with sat.
- 84
NaHCO3, diluted and extracted with DCM. The organic layer was separated and dried over sodium
sulfate. The organic solution was filtered and evaporated under reduced pressure to afford product
(1.77 (1.77 g). g).1H¹HNMR (500 NMR MHz,MHz, (500 Chloroform-d) 8 5.35 (tdt, Chloroform-d) J = 13.8, 5.35 (tdt, J =9.7, 5.09.7, 13.8, Hz, 4H), 5.0 4.84 (t, J 4.84 Hz, 4H), = 4.8 (t, J = 4.8
Hz, 1H), 4.24 (t, J = 7.0 Hz, 2H), 4.12 (q, J = 7.2 Hz, 3H), 3.96 (s, 2H), 3.90 - 3.77 (m, 2H), 3.00
(s, 3H), 2.77 (t, J = 6.8 Hz, 2H), 2.05 (d, J = 6.9 Hz, 9H), 1.77 - 1.59 (m, 5H), 1.52 - 1.15 (m,
28H), 0.89 (t, J = 6.8 Hz, 3H).
(13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)-N,N-dimethyldocosa-13,16-dien-1-amin (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)-N,N-dimethyldocosa-13,16-dien-1-amine.
N O O Chemical ChemicalFormula: Formula:C35H67NO2 CHNO Molecular Weight: 533.93
To a 150 mL pressure bottle, added a solution of (13Z,16Z)-3-(8-(1,3-dioxolan-2-
yl)octyl)docosa-13,16-dien-1-yl methanesulfonate (2.37 mmol, 1.39 g)in THF (20mL), added
dimethylamine (2M in THF, 14.22 mmol, 7.11 mL). The reaction mixture was sealed and heated
at 65 °C for overnight. The reaction mixture was cooled at 0 °C and quenched with brine, diluted
and extracted with EtOAc. The organic layer was separated and dried over sodium sulfate and
evaporated under vacuum to a yellow oil. The residue was purified by ISCO (SiO2: 30-100%
EtOAc/Hexane (3% trimethylamine added)) to provide product as a light yellow oil (1.38 g). 1H ¹H
NMR (400 MHz, Chloroform-d) 8 5.35 5.35 (qd, (qd, JJ == 10.9, 10.9, 5.3 5.3 Hz, Hz, 4H), 4H), 4.05 4.05 -- 3.76 3.76 (m, (m, 4H), 4H), 2.77 2.77 (t, (t, JJ
= 6.5 Hz, 2H), 2.20 (s, 6H), 2.05 (q, J = 6.9 Hz, 4H), 1.70 - 1.56 (m, 2H), 1.49 - 1.21 (m, 42H),
0.89 (t, J = 6.8 Hz, 3H).
(20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienal 0Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-diena
WO wo 2020/072324 PCT/US2019/053617
Chemical ChemicalFormula: Formula:C33H63NO CHNO Molecular Weight: 489.87
To a solution of (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)-N,N-dimethyldocosa-13,16-
dien-1-amine (2.58 mmol, 1.38 g) in acetone(10mL) was added 1N HCI (7 mL). The reaction
mixture was allowed to stir at room temperature overnight. TLC showed the completion of
reaction, to the reaction mixture slowly added 20% K2CO3 aqueoussolution K2CO aqueous solutionuntil untilPH~10, PH~10,diluted diluted
with EtOAc, the organic solution was extracted and separated, dried over sodium sulfate and
concentrated to a colorless oil. The residue was dried under high vacuum and yield title product
(1.1 g). 1H ¹H NMR (500 MHz, Chloroform-d) 8 9.70(s, 9.70(s, 1H), 1H), 5.36 5.36 (tdd, (tdd, JJ == 17.9, 17.9, 11.3, 11.3, 7.1 7.1 Hz, Hz, 4H), 4H),
2.77 (t, J = 6.9 Hz, 2H), 2.42 (td, J = 7.4, 1.9 Hz, 2H), 2.21 (s, 8H), 2.05 (q, J = 7.1 Hz, 4H), 1.63
(p, J = 7.4 Hz, 2H), 1.46 - 1.14 (m, 36H), 0.89 (t, J = 6.7 Hz, 3H).
(20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dien-1-ol (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dien-1-ol
HO Chemical Chemical Formula: C33H65NO Formula: CHNO Molecular Weight: 491.89
At 0 °C, to a solution of (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienal
(0.327 mmol, 160 mg) in anhydrous MeOH (4 mL) added NaBH4 (0.653 mmol, 25 mg). The
reaction mixture was allowed at stir at room temperature overnight. TLC showed completion of
reaction. The reaction mixture was quenched with sat NH4Cl, NH4CI, diluted with EtOAc and extracted.
The organic layer was dried over sodium sulfate and concentrated to colorless oil. The residue was
purified by ISCO (SiO2: 30-80% EtOAc/Hexane) to provide product 145 mg. 1H ¹H NMR (500 MHz,
Chloroform-d) 8 5.36 Chloroform-d) 5.36(dtd, J =J 17.9, (dtd, 11.2, = 17.9, 7.2 Hz, 11.2, 7.24H), Hz,4.12 4H),(q, J = (q, 4.12 7.2 Hz, J = 2H), 7.2 3.64 (t, J 3.64 Hz, 2H), = 6.6 (t, J = 6.6
Hz, 2H), 2.77 (t, J = 6.9 Hz, 2H), 2.21 (s, 8H), 2.05 (d, J = 6.5 Hz, 7H), 1.47 - 1.23 (m, 34H), 0.88
(tt, J =9.9,5.2Hz, = 6H). = 9.9, 5.2 Hz, 6H).
(20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dien-1-yl (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dien-1-j)l2-ethylhexanoate 2-ethylhexanoate
(ALNY-654)
Chemical ChemicalFormula: Formula:C41H79NO2 CHNO Molecular Weight: 618.09
654 654
To a 100 mL RBF, added a solution of (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-
20,23-dien-1-ol 20,23-dien-1-ol (0.295 mmol, (0.295 145 145 mmol, mg) in mg)anhydrous DCM (3 DCM in anhydrous mL),(3 to mL), this to added K2CO3 this (0.885 added KCO (0.885
mmol, 122 mg), cooled in an ice bath, added 2-ethylhexanoyl chloride (0.442 mmol, 71.9 mg).
The reaction mixture was allowed to stir at room temperature overnight. Quenched the reaction
with water, and extracted with DCM. The organic layer was separated and dried over sodium
sulfate. The combined organic solution was evaporated under reduced pressure. The residue was
purified by ISCO (SiO2: 30-80% EtOAc and Hexane (3% TEA added)) to provide product as a
colorless oil (78.2 mg). MS: M+1=618.7. 1HNMR ¹H NMR(500 (500MHz, MHz,Chloroform-d) Chloroform-d)S 5.36 (tdd, J = 17.8,
11.4, 7.1 Hz, 4H), 4.07 (t, J = 6.7 Hz, 2H), 2.78 (t, J = 6.9 Hz, 2H), 2.21 (s, 12H), 2.05 (q, J = 7.0
Hz, 5H), 1.44 - 1.14 (m, 43H), 0.88 (ddd, J = 7.3, 5.5, 3.3 Hz, 11H).
9-Bromononanal
Br Br
Chemical Formula: Chemical Formula:C9H17BrO CHBrO Molecular Weight: 221.14
- 87
To a 200mL RBF, added a suspension of pyridinium chlorochromate (30 mmol, 6.47 g)
and MgSO4 (2.5g) (2.5 g)in inDCM DCM(50 (50mL). mL).Cooled Cooledin inan anice icebath, bath,added addeda asolution solutionof of9-bromo-i-nonanol 9-bromo-i-nonanol
(20 mmoL, 4.46 g) in anhydrous DCM (10 mL) slowly. Allowed to stir at 0 °C for 1hr then rt for
1hr. Added diethylether (2x50 mL) to the reaction mixture and stirred vigorously for 5min, filtered
through celite. The residue was washed with 3x50mL of ether. The combined ether solution was
concentrated to product as a brown oil (4.06 g). 1H ¹H NMR (400 MHz, Chloroform-d) 8 9.76 9.76 (d, (d, JJ ==
1.8 Hz, 1H), 3.40 (t, J = 6.8 Hz, 3H), 2.43 (td, J = 7.3, 1.8 Hz, 2H), 1.84 (p, J = 6.9 Hz, 3H), 1.62
(p, J = 7.2 Hz, 3H), 1.42 (dq, J = 13.0, 6.9 Hz, 5H).
2-(8-bromooctyl)-1,3-dioxolane
Br Br
Chemical Chemical Formula: C11H21BrO2 Formula: CHBrO Molecular Weight: 265.19
To 250mL RBF, added a solution of 9-bromononanal (18.09 mmol, 4.0 g) in anhydrous
toluene (60 mL), added p-TsOH (0.946 mmol, 180 mg), followed by ethylene glycol (53.97
mmol, 3.35 g). The reaction was equipped with dean stark and condenser and refluxed at 130°C
overnight. The reaction mixture was quenched with sat. NaHCO3, extracted with EtOAc, washed
with brine. The organic solution was dried over sodium sulfate and evaporated under reduced
pressure to colorless oil. The residue was purified by ISCO (SiO2: 0-20% EtOAC/Hexane) to
provide product 3.12 g. 1H ¹H NMR (400 MHz, Chloroform-d) 8 4.84 4.84 (t, (t, JJ == 4.8 4.8 Hz, Hz, 1H), 1H), 4.07 4.07 -- 3.76 3.76
(m, 4H), 3.40 (t, J = 6.9 Hz, 2H), 1.84 (p, J = 7.0 Hz, 2H), 1.70 - 1.59 (m, 2H), 1.47 - 1.26 (m,
10H).
(8-(1,3-dioxolan-2-yl)octyl)magnesium bromide (8-(1,3-dioxolan-2-yl)octyl)magnesium bromide
MgBr
Chemical Formula: Chemical Formula:CHBrMgO BrMgO2 Molecular Weight: 289.50
88 -
To a 100mL 2-neck RBF, added pre-activated Mg-turnings (12.67 mmol, 308 mg) in THF
(16 mL), added a solution of 12-(8-bromooctyl)-1,3-dioxolane (6.34mmol, 2-(8-bromooctyl)-1,3-dioxolane (6.34 mmol,1.68 1.68g) g)in inTHF THFfollowed followed
by 1-2 crystals of Iodine. The reaction was allowed to stir at 60°C for 35min. The solution was
decolorized. Cooled to room temperature and used as is.
(20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoic acid (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoic : acid
/ Oxone N1 N1 DMF DMF HO Ho
0 C33H63NO C33H63NO2 CHNO Mol Wt: 489.87 CHNO Mol. Wt: 505.87
8 9
To a solution of (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienal (1.45
mmol, 711 mg) in anhydrous DMF (6 mL) was added Oxone (1.45 mmol, 892.3 mg). The reaction
mixture was allowed to stir at room temperature overnight. TLC showed completion of reaction,
water was added to quench the reaction, diluted and extracted with EtOAc, washed with brine. The
organic layer was separated and dried over sodium sulfate. The combined organic layer was
evaporated under reduced pressure. The residue was co-evaporated with toluene to remove any
water. Yield product (733 mg). MS: M+1=506.5.
(20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoyl chloride
Oxayl Chloride N N N CI HO O O C33H63NO2 C33H62CINO CHNO CHCINO Mol. Wt: 505.87 Mol. Wt: 524.32
10 9
- 89
At 0 °C, to a solution of (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoio (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoic.
acid (1.45 mmol, 733 mg) in anhydrous DCM (8 mL) was added 2 drops of anhydrous DMF
followed by neat oxayl chloride dropwise. The reaction mixture was stirred at room temperature
overnight. TLC showed completion of reaction (small sample of reaction mixture was treated with
MeOH). The reaction solution was concentrated to dark red oil.
3-pentyloctyl (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoate 3-pentyloctyl 20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoate
N N CI OH K2CO3 O O DCM DCM O C13H28O C33H62CINO CHCINO CHO Mol. Wt: 200.37 C46H89NO2 Mol. Wt: 524.32 CHNO Mol.Wt: 688.22 Mol.Wt 688.22 10 11 660
To a solution of(20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoyl of (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-dienoylchloride chloride
(0.944 mmol, 495 mg) in DCM(10 mL), K2CO3 (3.30mmol, K2CO (3.30 mmol,457 457mg) mg)was wasadded addedfollowed followedby byaa
solution of 3-pentyloctan-1-ol (1.89 mmol, 378.1 mg) in DCM(2 mL). The reaction mixture was
stirred at room temperature overnight. TLC showed completion of reaction. The reaction mixture
was quenched with water, diluted with DCM and extracted. The organic layer was separated and
dried over sodium sulfate and the organic solution was filtered and evaporated under reduced
pressure. The residue was purified by ISCO (SiO2: 0-40% EtOAc and Hexane) to provide product
(195.6 mg). (195.6 mg).MS: MS:M+1=689.7. 1H NMR M+1=689.7. (400 (400 ¹H NMR MHz, MHz, Chloroform-d) 8 5.35 (qd, Chloroform-d) 5.35J (qd, = 10.9, 5.6 J = Hz, 5.6 Hz, 10.9,
4H), 4.10 (dt, J = 20.3, 7.1 Hz, 4H), 2.77 (t, J = 6.5 Hz, 2H), 2.31 - 2.24 (m, 3H), 2.22 (s, 7H),
2.05 (d, J = 5.5 Hz, 6H), 1.44 - 1.15 (m, 55H), 0.89 (td, J = 7.0, 2.7 Hz, 9H).
2-Isopropyl-5-methylhexyl (20Z,23Z)-10-(2-(dimethylamino)ethyl)nonacosa-20,23-
dienoate
WO wo 2020/072324 PCT/US2019/053617
N1
Chemical ChemicalFormula: C43H83NO2 Formula: CHNO Molecular Weight: 646.14
661
¹H NMR (400 MHz, Chloroform-d) 8 MS: M+1=647.6. 1H 5.35 (tq, 5.35 J J (tq, = = 10.6, 6.3, 10.6, 5.4 6.3, Hz, 5.4 4H), Hz, 4H),
4.14 - 3.91 (m, 3H), 2.77 (t, J = 6.5 Hz, 2H), 2.29 (t, J = 7.5 Hz, 2H), 2.22 (s, 8H), 2.05 (q, J = 6.9 J=6.9
Hz, 4H), 1.77 (pd, J = 6.9, 4.7 Hz, 1H), 1.47-1.14 - (m, 42H), 0.88 (td, J = 6.2, 3.1 Hz, 17H). 1.47 - 1.14
1(20Z,23Z)-10-(2-(isopropyl(methyl)amino)ethyl)nonacosa-20,234 3-Pentyloctyl (20Z,23Z)-10-(2-(isopropyl(methyl)amino)ethyl)nonacosa-20,23-
dienoate dienoate
Chemical Chemical Formula: C48H93NO2 Formula: CHNO Molecular Weight: 716.28
662 662 MS: MS: M+1=717.8. M+1=717.8.1H¹H NMRNMR (400 MHz,MHz, (400 Chloroform-d) S 5.36 5.36 Chloroform-d) (dq, J(dq, = 12.4, J = 6.7, 5.66.7, 12.4, Hz, 5.6 Hz,
4H), 4.20 - 3.99 (m, 12H), 2.80 (dt, J = 18.0, 6.5 Hz, 2H), 2.30 (dt, J = 21.5, 7.6 Hz, 4H), 2.19 (s,
3H), 2.04 (s, 16H), 1.34 - 1.25 (m, 43H), 1.00 (d, J = 6.5 Hz, 5H), 0.88 (h, J = 2.9, 2.3 Hz, 9H).
2-Isopropyl-5-methylhexyl (20Z,23Z)-10-(2-(isopropyl(methyl)amino)ethyl)
nonacosa-20,23-dienoate nonacosa-20,23-dienoate
Y N 1
Chemical Chemical Formula: C45H87NO2 Formula: CHNO Molecular Weight: 674.20
663
- 91
WO wo 2020/072324 PCT/US2019/053617 PCT/US2019/053617
MS: MS: M+1=675.7. M+1=675.7.1H¹H NMRNMR (400 MHz,MHz, (400 Chloroform-d) 8 5.45 5.45 Chloroform-d) - 5.24- (m, 4H), 5.24 (m,4.12 (q,4.12 4H), J = (q, J =
7.2 Hz, 9H), 4.07 - 3.94 (m, 2H), 2.80 (dt, J = 21.8, 6.5 Hz, 3H), 2.31 (dt, J = 20.1, 7.7 Hz, 4H),
2.19 (s, 3H), 2.04 (s, 16H), 1.26 (t, J = 7.1 Hz, 21H), 1.00 (d, J = 6.5 Hz, 6H), 0.88 (ddt, J = 9.3,
6.3, 2.7 Hz, 19H).
6-steps 1) LAH
2) MsCl, MsCI, TEA OMs OMs
O O 1 2 2
KCN CN LAH LAH EtOH, H2O CN THF, 0C-rt NH2 O NH O 3 O 4
HCHO HCI N Na(OAc)3BH3 rt-o/n N MeOH O 5 6 6 Oxone N Oxyal Chloride DMF, rt r/n HO N O CI CI 7
OH 8
N K2CO3 O DCM
HO Ho N
(14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)tricosa-14,17-dienenitrile (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)tricosa-14,17-dienenitrile
- 92
WO wo 2020/072324 PCT/US2019/053617 PCT/US2019/053617
OMs KCN CN O O EtOH, H2O O O C34H64O5S C34H61NO2 CHOS Mol Wt: 584.94 CHNO Mol Wt: 515.87
6 7
To a solution of (13Z,16Z)-3-(8-(1,3-dioxolan-2-yl)octyl)docosa-13,16-dien-1-yl
methanesulfonate (5.13 mmol, 3 g) in EtOH (200 proof) was added aqueous solution of
KCN(15.39 mmol, KCN(15.39 mmol, 1.02 1.02 g), g), the the reaction reaction was was refluxed refluxed at at 80 80 °C °C overnight. overnight. The The condenser condenser was was
connected to a KOH trapper to neutralize the HCN released. The reaction mixture was quenched
with sat. NaHCO3, extracted with ether, washed with brine. The organic layer was dried over
sodium sulfate and filtered. The organic solution was evaporated under reduced pressure to a
colorless oil. The residue was purified by ISCO (SiO2: 0-20% EtOAc/Hexane) to provide product
(2.08 g). MS: ES*(M-1=514.3). ES'(M-1=514.3). 1H ¹H NMR (400 MHz, Chloroform-d) 85.34 5.34(ddd, (ddd,JJ==17.5, 17.5,11.3, 11.3,
6.6 Hz, 4H), 4.84 (t, J=4.8Hz, 1H), J = 4.8 Hz, 4.05 1H), - 3.73 4.05 (m, (m, - 3.73 4H), 2.77 4H), (t, (t, 2.77 J = J6.5 Hz, Hz, = 6.5 2H), 2.31 2H), (t, (t, 2.31 J = J7.6 = 7.6
Hz, 2H), Hz, 2H),2.14 - 1.93 (m, 2.14-1.93 (m,4H), 4H),1.70 - 1.57 1.70 (m, (m, - 1.57 4H), 4H), 1.56 1.56 (s, 2H), (s,1.47 2H),- 1.47 1.18 (m, 34H),(m, - 1.18 0.8934H), (t, J 0.89 = (t, J =
6.8 Hz, 3H).
(14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)tricosa-14,17-dien-1-amine (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)tricosa-14,17-dien-1-amine
CN LAH O NH2 THF O NH O O.
C34H61NO2 C34H65NO2 CHNO Mol Wt: 515.87 CHNO Mol. Wt: 519.90
7 8
A solution of (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)tricosa-14,17-dienenitrile (4.03
mmol, 2.08 g) in anhydrous THF at 0 °C, added 1N lithium aluminum hydrate in THF (8.06 mmol,
8.06 mL). The reaction mixture was stirred at 0 °C for 25min then at room temperature for 4 hours.
Cooled in an ice bath, to the reaction mixture was added a sat. sodium potassium tartrate
tetrahydrate solution dropwise until no bubbling and a ppt was formed. The ppt was filtered
-93- - - through celite. The solution was extracted with EtoAC and brine. The organic layer was separated and dried over sodium sulfate and filtered. The organic solution was evaporated under reduced pressure to a colorless oil. The residue was dried in the high vacuum overnight yield 2 g product.
MS: M+1=520.5. 1H ¹H NMR (400 MHz, Chloroform-d) 85.35 5.35(qd, (qd,JJ==10.8, 10.8,5.3 5.3Hz, Hz,4H), 4H),4.84 4.84(t, (t,JJ
= 4.9 Hz, 1H), 4.04 - 3.80 (m, 4H), 2.77 (t, J = 6.5 Hz, 2H), 2.66 (t, J = 7.1 Hz, 1H), 2.11 - 1.98
(m, 4H), 1.71 - 1.59 (m, 3H), 1.47 - 1.06 (m, 43H), 0.89 (t, J = 6.8 Hz, 3H).
14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)-N,N-dimethyltricosa-14,17-dien-1-amin (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)-N,N-dimethyltricosa-14,17-dien-1-amine
HCHO NH2 NH Na(OAc)3BH3 Na(OAc)3BH N O O C34H65NO2 CHNO Mol. Wt: 519.90 C36H69NO2 CHNO Mol. Wt: 547.95
8 9 9
To a solution of (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)tricosa-14,17-dien-1-amine
(3.85 mmol, 2 g) in anhydrous MeOH (20 mL) was added formal aldehyde (30% in water, 10 mL)
followed by Na(OAC)3BH3(15.39 mmol, 3.26 g). The reaction mixture was stirred at room
temperature overnight. TLC showed completion of reaction. The reaction mixture was quenched
with 1N NaOH, extracted with EtOAc, washed with brine. The combined organic layer was dried
over sodium sulfate and filtered. The organic solution was evaporated under reduced pressure to a
colorless oil. The residue was purified by ISCO (SiO2, 0-80% EtOAc/Hexane) to provide product
1H NMR (400 MHz, Chloroform-d) S5.49 (2.3 g). MS: M+1=548.5. ¹H 5.49--5.23 5.23(m, (m,4H), 4H),4.96 4.96--4.67 4.67
(m, 2H), 4.03 - 3.90 (m, 2H), 3.90 - 3.76 (m, 2H), 3.39 (s, 1H), 2.77 (t, J = 6.5 Hz, 2H), 2.25 (s,
1H), 2.16 (s, 1H), 2.11 (s, 1H), 2.09 - 1.99 (m, 5H), 1.78 (s, 1H), 1.69 - 1.61 (m, 2H), 1.59 (s,
5H), 1.46 - 1.14 (m, 37H), 0.89 (t, J = 6.8 Hz, 3H).
(20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienal
WO wo 2020/072324 PCT/US2019/053617
HCI N N N O Acetone O C36H6gNO2 CHNO Mol. Wt: 547.95 C34H65NO CHNO Mol. Wt: 503.90
9 10
To a solution of (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octy1)-N,N-dimethyltricosa-14,17 (14Z,17Z)-4-(8-(1,3-dioxolan-2-yl)octyl)-N,N-dimethyltricosa-14,17-
dien-1-amine (3.65 mmol, 2.3 g) in Acetone (25 mL), added 1N HCI HCl (10 mL). The reaction
mixture was stirred at room temperature overnight. The reaction mixture was quenched with 20%
K2CO3 aqueous KCO aqueous solution, solution, extracted extracted with with EtOAc, EtOAc, washed washed with with brine. brine. The The organic organic layer layer was was dried dried
over sodium sulfate and filtered. The organic solution was evaporated under reduced pressure to aa
colorless oil. Yield product 1.88 g. 1H ¹H NMR (400 MHz, Chloroform-d) 8 9.76 9.76 (d, (d, JJ == 1.9 1.9 Hz, Hz, 1H), 1H),
5.36 (tq, J = 11.0, 5.6, 4.3 Hz, 4H), 4.12 (q, J = 7.1 Hz, 1H), 2.77 (t, J = 6.5 Hz, 2H), 2.41 (td, J =
7.4, 1.9 Hz, 2H), 2.22 (s, 4H), 2.10-1.99 - (m, 6H), 1.68 - 1.55 (m, 2H), 1.47 - 1.10 (m, 40H), 2.10 - 1.99
0.89 (t, 0.89 (t,J J= =6.8 8 Hz, Hz, 3H). 3H).
(20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoic : acid acid (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoic
Oxone N N N DMF HO Ho O C34H65NG CHNO Mol. Wt: 503.90 C34H65NO2 CHNO Mol.Wt: 519.90
10 11
To a solution of (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienal (3.78
mmol, 1.88 g) in anhydrous DMF(15 mL) was added Oxone(3.72 mmol, 2.29 g). The reaction
mixture was stirred at room temperature overnight. TLC showed completion of reaction. The
reaction mixture was quenched with brine, diluted with EtOAc and extracted. The organic layer
was separated and dried over sodium sulfate and filtered. The organic solution was evaporated
under reduced pressure to a colorless oil. The residue was co-evaporated with toluene 2x20mL to
remove any trace of water. Yield 1.99 g product. MS: M+1=520.5
-95-
(20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoylchloride (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoyl chloride
Oxayl Chloride N N N CI Ho HO
C34H65NO2 C34H64CINO CHNO CHCINO Mol.Wt Mol.Wt:519.90 519.90 Mol. Wt: 538.34
11 12
To a solution of (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoic acid
(3.82 mmol, 1.99 g) in anhydrous DCM at 0°C, added 2 drops of anhydrous DMF followed by
oxayl chloride(9.57 mmol, 833 uL) slowly. The reaction mixture was stirred at room temperature
overnight. TLC showed completion of reaction. The reaction mixture was concentrated to provide
product as brownish red oil.
3-pentyloctyl (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoate 3-pentyloctyl (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoate
OH N K2CO3 N CI CI O DCM C13H28O C34H64CINO CHCINO CHO Mol. Wt: 200.37 C47H91NO2 Mol. Wt: 538.34 CHNO Mol. Mol. Wt: Wt: 702.25 702.25 12 13 664
To a solution of (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-dienoyl
chloride (2.22 mmol, 1.2 g) in anhydrous DCM(10 mL), added K2CO3 (7.77 mmol, K2CO (7.77 mmol, 1.07 1.07 g) g)
followed by a solution of 3-pentyloctan-1-ol (4.46 mmol, 0.893 g)in anhydrous DCM. The
reaction mixture was stirred at room temperature overnight. TLC showed completion of reaction.
The reaction mixture was quenched with brine, and diluted with DCM. The organic layer was
extracted, separated, and dried over sodium sulfate. The organic solution was filtered and
evaporated under reduced pressure. The residue was purified by ISCO (SiO2: 0-40% EtOAc (SiO: 0-40% EtOAc and and
¹H NMR (400 MHz, hexane) to provide product as a brown-red oil (99.5 mg). MS: M+1=703.7. 1H
Chloroform-d) 8 5.36 5.36 (tq, (tq, JJ == 10.9, 10.9, 5.5, 5.5, 4.2 4.2 Hz, Hz, 4H), 4H), 4.08 4.08 (t, (t, JJ == 7.1 7.1 Hz, Hz, 2H), 2H), 2.77 2.77 (t, (t, JJ == 6.5 6.5 Hz, Hz,
-96-
2H), 2.28 (t, J = 7.5 Hz, 2H), 2.21 (s, 6H), 2.05 (q, J = 6.9 Hz, 4H), 1.57 (q, J = 6.8 Hz, 10H), 1.44
- 1.14 (m, 52H), 0.89 (td, J = 6.9, 2.6 Hz, 8H).
2-Isopropyl-5-methylhexyl (20Z,23Z)-10-(3-(dimethylamino)propyl)nonacosa-20,23-
dienoate
Chemical Chemical Formula: C44H85NO2 Formula: CHNO Molecular Weight: 660.17
665 MS: M+1=661.7. 1H ¹H NMR (400 MHz, Chloroform-d) 8 5.35 5.35 (tq, (tq, J J = = 11.1, 11.1, 7.0, 7.0, 5.6 5.6 Hz, Hz, 4H), 4H),
4.15 - 3.88 (m, 2H), 2.77 (t, J = 6.5 Hz, 2H), 2.42 - 2.16 (m, 9H), 2.05 (q, J = 6.9 Hz, 4H), 1.77
(pd, J = 6.9, 4.6 Hz, 1H), 1.61 (p, J = 7.1 Hz, 2H), 1.53 - 1.03 (m, 46H), 0.88 (td, J = 6.2, 3.1 Hz,
14H).
Example 3: Preparation of Lipid Nanoparticles
The cationic lipids described herein are used to formulate liposomes containing the
AD-1661 duplex (shown in the table below) using an in-line mixing method as described in
International Publication No. WO 2010/088537, which is incorporated by reference in its entirety.
Unless indicated Unless indicated otherwise, otherwise, the lipid the lipid nanoparticles nanoparticles had the had the formulation formulation shown shown in the inbelow. table the table below.
wo 2020/072324 WO PCT/US2019/053617 PCT/US2019/053617
Component Mole Percentage (Based on 100% of the lipid components components in in the the LNP) LNP)
Cationic lipid 50% Distearoylphosphatidylcholine (DSPC) 10% Cholesterol 38.5% 38.5% 1-(monomethoxy-polyethyleneglycol)- 1-(monomethoxy-polyethyleneglycol)- 1.5% 2,3-dimyristoylglycerol (PEG-DMG) (with an average PEG molecular weight of 2000)
siRNA (AD-1661) --
Formulations having a formulation of "58/10/30/2" had the formulation shown in the table
below.
Component Mole Percentage (Based on 100% of the lipid components in the LNP)
Cationic lipid 58%
DSPC 10% Cholesterol 30% PEG-DMG (with an average PEG 2% molecular weight of 2000)
siRNA (AD-1661) -
Formulations having a formulation of "55/10/33/2" had the formulation shown in the table
below.
PCT/US2019/053617
Component Mole Percentage (Based on 100% of the lipid components in the LNP)
Cationic lipid 55%
DSPC 10% Cholesterol 33% PEG-DMG (with an average PEG 2% molecular weight of 2000)
siRNA (AD-1661) -
Formulations having a formulation of "50/10/38/2" contain 50 mol % cationic lipid, 10
mol % DSPC, 38 mol % cholesterol, and 2 mol % PEG-DMG.
The siRNA AD-1661 duplex has the sequence shown below.
Duplex Sequence 5'-3' SEQ Target ID NO: 1 AD-1661 GGAfUfCAfUfCfUfCAAGfUfCfUfUAfCdTsd GGAfUfCAfUfCfUfCAAGfUfCfUfUAfCdTsdT FVII
GfUAAGAfCfUfUGAGAfUGAfUfCfCdTsdT 2
Lower case is 2'OMe modification and Nf is a 2'F modified nucleobase, dT is
deoxythymidine, S is phosphothioate
The lipid nanoparticles were prepared as follows. Cationic lipid, DSPC, cholesterol, and
PEG-DMG in the ratio recited in the table above were solubilized in ethanol at a total lipid
concentration of 25 mg/mL.
A siRNA stock solution was prepared by solubilizing the siRNA AD-1661 in a low pH
acetate or citrate buffer (pH=4) at 0.8 mg/mL.
The stock solutions should be completely clear and the lipids should be completely
solubilized before combining with the siRNA. Therefore, if it was determined appropriate, the
stock solutions were heated to completely solubilize the lipids.
WO wo 2020/072324 PCT/US2019/053617
The individual stock solutions were combined by pumping each solution to a T-junction
(i.e., by in-line mixing). Specifically, the ethanol solution (at 5 ml/min, via 0.01 in. PEEK tube)
and aqueous buffer solution (at 15 mL/min, via 0.02 in. PEEK tube) were mixed through a T-
junction (PEEK Tee body, IDEX).
After the T-junction a single tubing is placed where the combined stream will emit. Ethanol
is removed and exchanged for PBS by dialysis. The lipid formulations are then concentrated using
centrifugation or diafiltration to an appropriate working concentration.
Lipid nanoparticles containing the cationic lipids listed in the table in Example 36 were
prepared as described above.
Example 4: Efficacy of Lipid Nanoparticles
Factor VII (FVII), a prominent protein in the coagulation cascade, is synthesized in the
liver (hepatocytes) and secreted into the plasma. FVII levels in plasma can be determined by a
simple, plate-based colorimetric assay. As such, FVII represents a convenient model for
determining siRNA-mediated downregulation of hepatocyte-derived proteins.
Test formulations of the lipid nanoparticles prepared in Example 3 were assessed for their
FVII knockdown in female 7-9 week old, 15-25g, female C57B1/6 mice at 0.01 and 0.03 mg/kg
with 3 mice per treatment group. All studies included animals receiving either phosphate-buffered
saline (PBS, control group) or a benchmark formulation. Formulations were diluted to the
appropriate concentration in PBS immediately prior to testing. Mice were weighed and the
appropriate dosing volumes calculated (10 ul/g µl/g bodyweight). Test and benchmark formulations
as well as PBS (for control animals) were administered intravenously via the lateral tail
vein. Animals were anesthetised 24 hours later with an intraperitoneal injection of
ketamine/xylazine and 500-700 ul µl of blood was collected by cardiac puncture into serum separator
tubes (BD Microtainer). Blood was centrifuged at 2,000 X x g for 10 minutes at 15° C and serum
was collected and stored at -70° C until analysis. Serum samples were thawed at 37° C for 30
minutes, diluted in PBS and aliquoted into 96-well assay plates. Factor VII levels were assessed
- 100 wo 2020/072324 WO PCT/US2019/053617 using a chromogenic assay (Biophen FVII kit, Hyphen BioMed) according to the manufacturer's instructions and absorbance was measured in a microplate reader equipped with a 405 nm wavelength filter.
The efficacy of lipid nanoparticle formulations containing the cationic lipids below was
determined by the procedure above. Figure 1 shows the relative FVII protein levels at day 3 (at
an siRNA concentration of 0.01 and 0.03 mg/kg). Formulation AF-011 contained the cationic
lipid known as MC3.
Formulation Formulation Cationic Lipid
AF-011 O AD-1661 N
AF-060 O AD-1661 O N / AF-062 N AD-1661
O AF-064 O AD-1661
AF-065 N AD-1661
The efficacy of lipid nanoparticle formulations containing the cationic lipids below was
determined by the procedure above. Figure 2 shows the relative FVII protein levels at day 2 (at
an siRNA concentration of 0.01 and 0.03 mg/kg). The logP values for the cationic lipids listed in
WO wo 2020/072324 PCT/US2019/053617
the table calculated using software available the table below were below werecalculated using thethe software available at at
http://www.molinspiration.com/services/logp.htm http://www.molinspiration.com/services/logp.html from Molinspiration from Molinspiration Cheminformatics Cheminformatics of of
Slovensky Grob, Slovak Republic.
Formulation Cationic Lipid Formulation Formulation Log P
Ratio / N/P
Ratio
AF-011 50/10/38.5/1.5 10.201 O AD-1661 N N/P =2.97 O AF-068 O 58/10/30/2 10.42 AD-1661 N/P =2.97 O N O O AF-054* AF-054* O O 50/10/38.5/1.5 10.313 AD-1661 N/P =3.01 0 O O N O
AF-069 58/10/30/2 9.94 N N/P =3.01 AD-1661
AF-070 58/10/30/2 10.313 O AD-1661 N/P =2.97 O O O N
50/10/38.5/1.5 10.43 AF-071 AF-071 O O N/P =2.97 AD-1661 N
AF-072 50/10/38.5/1.5 10.28
AD-1661 O 0 N/P =2.97
AF-073 58/10/30/2 10.43 AD-1661 N/P =2.97 O
AF-074 58/10/30/2 10.28 AD-1661 N/P =2.97
AF-075 50/10/38.5/1.5 10.48 AD-1661 N/P =2.97
AF-076 50/10/38.5/1.5 10.35 O N/P =3.01 AD-1661
AF-077 58/10/30/2 10.48
AD-1661 N/P =2.97
AF-078 58/10/30/2 10.28 AD-1661 N/P =2.97
* PEG-DPG (1,2-dipalmityl-sn-glycerol-methoxy (1,2-dipalmityl-sn-glycerol-methoxy.polyethylene polyethyleneglycol) glycol)was wasused usedinstead insteadof ofPEG- PEG-
DMG. The efficacy of lipid nanoparticle formulations containing the cationic lipids below was
determined by the procedure above. Figure 3 shows the relative FVII protein levels (at a siRNA
concentration of 0.01 and 0.03 mg/kg). N/P refers to the ratio of amino groups of the cationic
lipids to phosphate groups in the siRNA.
Formulation Cationic Lipid Formulation Formulation N/P Ratio
Ratio
AF-011 AF-011 50/10/38.5/1.5 3.0 N AD-167990 O
AF-079 50/10/38/2 2.628
AD-167990
AF-093 55/10/33/2 3.0
AD-167990
AF-083 55/10/33/2 3.76 O AD-167990
AF-073 58/10/30/2 3.03
O O AD-167990
AF-094 50/10/38/2 50/10/38/2 3.0 O AD-167990 O O O N O
The structure of AD-167990 is shown below.
Sequence 5'-3' SEQ Target Duplex ID NO:
AD-167990 gsasaacuCfaAfUfAfaagugcuuusa 3 FXII
usAfsaagCfacuuuauUfgAfguuucususg usAfsaagCfacuuuauUfgAfguuucususg 4
Lower case is 2'OMe modification and Nf is a 2'F modified nucleobase, dT is
deoxythymidine, S is phosphothioate
The efficacy of lipid nanoparticle formulations containing the cationic lipids below was
determined by the procedure above. Figure 4 shows the relative FVII protein levels after 48 hours
(at a siRNA concentration of 0.005, 0.01, and 0.03 mg/kg).
Formulation Cationic Lipid Formulation Formulation Ratio / N/P Ratio
AF-070 O 58:10:30:2 AD-1661 O N/P=3 N
AF-068 O 58:10:30:2 AD-1661 O N/P=3 O N
AF-072 50/10/38.5/1.5
AD-1661 O N/P=3
AF-073 58:10:30:2
AD-1661 N/P=3
AF-074 58/10/30/2 AD-1661 O O N/P=3
Example 5: Non-Human Primate Study
The purpose of this study is to determine the pharmacodynamics of multiple Lipid Nano
Particles (LNPs) targeting F12 protein after a single intravenous infusion dose to male and/or female
cynomolgus monkeys. Five lipids nanoparticle formulations with different lipids (AF-011, AF-070,
AF-079, AF-073 and AF-074) having F12 siRNA AD-167990 were tested in this study. Each of these
WO wo 2020/072324 PCT/US2019/053617
formulations were administered to cynomolgus monkeys on Day 1 by intravenous infusion over
approximately 60 minutes (3 animals/group/formulation) with a targeted dose of 0.3 mg/kg. For all
groups, blood (approximately 1 mL) were collected from each animal on Days -5, -1, 1 (predose), 2,
3, 5, 8, 15, 22, 29, 36, 43, 50, 57, and 64. F12 plasma protein levels were determined using F12 assay
using human factor XII total antigen assay ELISA kit from Molecular Innovations (HFXIIKT-TOT).
The formulations described in the table below were prepared as described in Example 3
with the F12siRNA AD-167990.
Formulation Cationic Lipid Formulation N/P Ratio
Ratio
AF-011 AF-011 50/10/38.5/1.5 3 O N AD-167990 O
AF-070 58/10/30/2 2.628 O AD-167990 O O O N O O AF-079 50/10/38/2 2.628 O AD-167990 O O O N O AF-073 58/10/30/2 3.03
O AD-167990
AF-074 58/10/30/2 3.23
O O O AD-167990
The table below provides the protocol used for each formulation. Figure 5 shows the
relative F12 plasma levels (relative to pre-dose).
Formulation Group Dose Level Route Route and and N Number Number (mg/kg) Regimen 1 0.3 60 minutes AF-011(MC3)-AD AF-011(MC3)-AD- 4(2F=2M) 167990 IV Infusion
AF-070(ALNY369)-AD- 2 3(3M) 3(3M) 0.3 60 minutes
167990 IV Infusion
AF-079(ALNY369)-AD- 3 3(3F) 0.3 60 minutes
167990 IV Infusion
AF-074(ALNY369)-AD- 4 3(3F) 0.3 60 minutes
167990 IV Infusion
AF-073(ALNY369)-AD- 5 3(3M) 3(3M) 0.3 60 minutes
167990 IV Infusion
Example 6: Non-Human Primate Study
This was a single-dose pharmacodynamic (PD) / pharmacokinetic (PK) study of multiple Lipid
Nano Particles (LNPs) targeting Factor XII (F12) protein after a single intravenous infusion dose to
male and/or female cynomolgus monkeys. Six lipids nanoparticle formulations with different lipids
(AF-079, AF-093, AF-0783, AF-073, AF-094 and AF-011) having F12 siRNA AD-167990 were tested
in this study. Each of these formulations were administered to cynomolgus monkeys on Day 1 by
intravenous infusion over approximately 60 minutes (3 animals/group/formulation) with a targeted
dose of 0.03, 0.1 or 0.3 mg/kg. F12 plasma protein levels were determined using a F12 assay using a
- 107
WO wo 2020/072324 PCT/US2019/053617 PCT/US2019/053617
human factor XII total antigen assay ELISA kit from Molecular Innovations (HFXIIKT-TOT) as
reported earlier.
The study design is shown in the table below.
Dose Level Dose Volume Concentration Collection Number (mg/mL) B B Group Test Article of Males Dose Route Vehicle (mg/kg) (mg/kg) (mL/kg) (mg/mL) Intervals C.D 1 AD-167990.1 in AF-011 3 IV Infusion A 0.3 10 0.030 Blood Blood 10 10 C,D 2 AD-167990.1 in AF-094 3 3 IV Infusion A 0.03 0.003 Blood Blood 10 C,D 3 AD-167990.1 in AF-094 3 3 IV Infusion A 0.1 10 0.010 Blood Blood 10 C,D 4 AD-167990.1 in AF-094 3 IV Infusion A 0.3 0.030 Blood 10 C.D 5 AD-167990.1 in AF-079 3 3 IV Infusion A 0.3 10 0.030 Blood Blood 10 C.D 6 AD-167990.1 in AF-073 3 IV Infusion A 0.03 10 0.003 Blood Blood 10 10 C,D 7 AD-167990.1 in AF-073 3 3 IV Infusion A 0.1 0.010 Blood Blood 10 C.D 8 AD-167990.1 in AF-073 3 IV Infusion A 0.3 0.3 10 0.030 Blood A 10 10 9 AD-167990.1 in AF-093 3 IV Infusion 0.03 0.003 Blood Blood A 10 10 10 AD-167990.1 in AF-093 3 IV Infusion 0.1 0.1 0.010 Blood Blood 10 C,D C.D 11 11 AD-167990.1 in AF-093 3 IV Infusion A 0.3 0.3 0.030 Blood 10 10 C,D 12 AD-167990.1 in AF-083 3 3 IV Infusion A 0.1 0.010 Blood Blood 10 C,D 13 AD-167990.1 in AF-083 3 IV Infusion A 0.3 0.030 Blood Blood A A 0.9% Sodium Chloride B B Dosed based on siRNA concentration C PK: Blood samples will be collected predose and at 0 (just prior to the end of infusion), 0.033, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, 168, 336,
672 and 1344 hours postdose. Sample collection times will be based off the end of infusion. D D PD: Blood samples will be collected on Days -5, -1, predose and at 24, 48, 96, 168, 336, 504, 672, 840, 1008, 1176, 1344 and 1512 hours hours postdose. postdose. Sample Sample collection collection times times will will be be based based off off the the end end of of infusion. infusion.
The formulations described in the table below were prepared as described in Example 3
with the F12siRNA AD-167990.
Formulation Cationic Lipid Formulation N/P Ratio Ratio
AF-0079 50/10/38/2 6 O AD-167990 O O O N
WO wo 2020/072324 PCT/US2019/053617
AF-093 55/10/33/2 6
AD-167990 AD-167990 O O
AF-083 55/10/33/2 7 7
AD-167990 O O
AF-073 58/10/30/2 6
AD-167990 O O O
AF-094 58/10/30/2 7 O AD-167990 AD-167990 O O N O
AF-011 AF-011 58/38.5/10/1.5 5.71
AD-167990 O N O
Figures 6A, 6B, and 7-9 show the dose response with AF-094, a single dose of AF-079,
AF-073, AF-093, and AF-083, respectively.
All references cited herein are incorporated by reference.
Throughout thisspecification specification and and the the claims whichfollow, follow,unless unless the the context context requires requires 01 Apr 2025 2019351809 01 Apr 2025
Throughout this claims which
otherwise, otherwise, the the word "comprise",and word "comprise", andvariations variationssuch suchasas "comprises" "comprises"oror"comprising", "comprising",will willbebe understood to imply the inclusion of a stated integer or step or group of integers or steps but understood to imply the inclusion of a stated integer or step or group of integers or steps but
not the exclusion of any other integer or step or group of integers or steps. not the exclusion of any other integer or step or group of integers or steps.
The reference in this specification to any prior publication (or information derived The reference in this specification to any prior publication (or information derived
from it), or from it), or to to any anymatter matterwhich which is known, is known, is and is not, not,should and should not be not be taken as taken an as an 2019351809
acknowledgment acknowledgment or or admission admission or any or any form form of suggestion of suggestion thatthat that that priorpublication prior publication(or (or information derived from information derived fromit) it) or or known matterforms known matter formspart partofofthe the common common general general knowledge knowledge
in in the field of the field of endeavour endeavour to to which which this this specification specification relates. relates.
- 109A 109A --
THE CLAIMS DEFINING THE THE INVENTION INVENTION ARE ARE AS AS FOLLOWS: 2019351809 01 Apr 2025
THE CLAIMS DEFINING FOLLOWS: 1. 1. A compound A compound of of formula formula (A): (A):
Z¹ R¹ R R X * N a b Q M R'
R² Z² 2019351809
Formula (A) Formula (A)
or a salt or a salt thereof, wherein thereof, wherein
R’ is absent, R' is absent,hydrogen, hydrogen, or alkyl; or alkyl;
with respect to R and R2, 1 R², with respect to R¹ and
(i) R1 and (i) R¹ andR²R2are areeach, each,independently, independently, optionally optionally substituted substituted alkyl,alkenyl, alkyl, alkenyl, alkynyl, cycloalkylalkyl, alkynyl, cycloalkylalkyl, heterocycle, heterocycle, or R10; or R¹;
(ii) R¹1 and (ii) R R2,together andR², together with with thethe nitrogen nitrogen atom atom to which to which they arethey are attached, attached, form form an optionallysubstituted an optionally substituted heterocylic heterocylic ring;ring; or or
(iii) (iii)one one of R¹1 and of R 2 an optionally substituted alkyl, alkenyl, alkynyl, andR²Ris is an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkyl, cycloalkylalkyl, or orheterocycle, heterocycle,and andthe theother otherforms formsa a4-10 4-10membered heterocyclic membered heterocyclic
ring or ring or heteroaryl heteroaryl with with (a) (a) the the adjacent adjacent nitrogen nitrogen atom and(b) atom and (b) the the (R)a (R)a group groupadjacent adjacenttotothe the nitrogen atom; nitrogen atom;
each occurrenceofof RRis, each occurrence is, independently, –(CR3R4)-; independently, -(CR³R)-;
each each occurrence R3and occurrenceofof R³ 4 andRRare, are,independently independentlyH, H, halogen, halogen, OH,OH, alkyl, alkyl, alkoxy, alkoxy, -NH2, -NH,
R10,alkylamino, R¹, alkylamino,orordialkylamino; dialkylamino;
10 independently selected from PEG and polymers based on each occurrenceofofR¹Ris each occurrence is independently selected from PEG and polymers based on poly(oxazoline), poly(ethylene poly(oxazoline), oxide), poly(vinyl poly(ethylene oxide), poly(vinyl alcohol), alcohol), poly(glycerol), poly(glycerol), poly(N- poly(N- vinylpyrrolidone), vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] poly[N-(2-hydroxypropyl)methacrylamide] and and poly(amino poly(amino acid)s, acid)s, wherein wherein
(i) (i) the the PEG or polymer PEG or polymerisislinear linearororbranched, branched,(ii) (ii) the the PEG PEGor or polymer polymer is polymerized is polymerized by n by n
subunits, subunits, (iii) (iii)n is a number-averaged n is a number-averageddegree degree of ofpolymerization polymerization between 10and between 10 and200 200units, units, and and 10 (iv) (iv) the thecompound hasatat most compound has mosttwo twoR¹Rgroups; groups;
-- 110 the dashed line to Q is absent or a bond; 01 Apr 2025 2019351809 01 Apr 2025 the dashed line to Q is absent or a bond; whenthe when thedashed dashedline linetotoQ Qisisabsent absentthen thenQ Q is is absentororisis-0-, absent -O-,-NH-, -NH-, -N(R5-S-, -N(R)-, )-, -S-, - - C(O)-, -C(O)O-, C(O)-, -C(0)0-, -OC(O)-, -C(O)N(R4)-, -OC(O)-, -C(O)N(R)-, -N(R5)C(O)-, -N(R)C(O)-, -S-S-, -S-S-, -OC(O)O-, -0C(0)0-, -O-N=C(R5)-, -O-N=C(R)-, - 5 C(R C(R)=N-O-, -OC(O)N(R5-N(R°)C(O)N(R³)-, )=N-O-,-OC(O)N(R)-, )-, -N(R5)C(O)N(R-N(R°)C(O)O-, 5 )-, -N(R5)C(O)O-, -C(O)S-, -C(O)S-, -C(S)O- -C(S)O- or - or - 5 C(R )=N-O-C(O)-;or C(R²)=N-O-C(O)-; or 2019351809 whenthe when thedashed dashedline linetotoQ Qisisa abond bondthen then(i)(i)b bisis 00 and and(ii) (ii) Q andthe Q and thetertiary tertiary carbon carbon adjacent adjacent totoitit(C*) (C*)form form a substituted a substituted or unsubstituted, or unsubstituted, mono- mono- or or bi-cyclic bi-cyclic heterocyclic heterocyclic group group having from having from55toto 10 10 ring ring atoms; atoms;
5 each occurrenceofof RRis, each occurrence is, independently, independently,HHororalkyl; alkyl;
X is alkylene or alkenylene; X is alkylene or alkenylene;
M¹ 1is M is aa biodegradable group; biodegradable group;
aa is is 1,1, 2,2,3,3,4,4,5 5or or 6; 6;
b is 0, 1, 2, or 3; b is 0, 1, 2, or 3;
Z1 is Z¹ is aaCC-C 6-Cbranched 14 branched alkyl alkyl group; group; andand
Z2 is Z² is CC-C 4-Calkenyl, 20 alkenyl, wherein wherein thethe alkenyl alkenyl group group maymay optionally optionally be substituted be substituted with with oneone
or or two fluorine atoms two fluorine at the atoms at the alpha alpha position positiontotoa a double doublebond bond which which is isbetween between the the double bond double bond
and theterminus and the terminus Z².Z2. of of
1 2 2. 2. The compound The compoundofof claim claim 1, 1, wherein whereinR’R R N-(R)a-Q-(R)b- isis(CH R'R¹R²N-(R)a-Q-(R)- 3)2N-(CH2)2-, (CH)N-(CH)-,
(CH3)2N-(CH2)3-C(O)O-, (CH)N-(CH)-NH-C(O)O-, (CH)N-(CH)-C(O)O-, (CH3)2N-(CH2)2-NH-C(O)O-,(CH)N-(CH)-OC(O)-NH-, (CH3)2N-(CH2)2-OC(O)-NH-, or or (CH3)2N-(CH2)3-C(CH3)=N-O-. (CH)N-(CH)-C(CH)=N-O-
1 R² are 3. 3. The compound The compoundof of claim claim 1, 1, wherein wherein R¹ R andand R2 are both both alkyl. alkyl.
4. 4. The compound The compoundof of claim claim 1 or 1 or 2, 2, wherein wherein is1 -OC(O)- M¹ M is -OC(O)- or -C(O)O-. or -C(0)0-.
1 5. 5. The compound The compoundofofany anyone oneof of claims claims 11 to to 4, 4,wherein whereinZZ¹isis a Ca6-C C-C branched 10 branched
alkyl group alkyl group
-- 111
6. The compound compound of of any any one one ofof claims claims 11 toto 4,4, wherein Z1 is wherein Z¹ is -- 30 May 2025 2019351809 30 May 2025
6. The i i CH(CH2CH3)(CH2CH2CH2CH CH(CHCH)(CHCHCHCH), 3), –CH2CH( Pr)(CH2CHor -CHCH('Pr)(CHCH'Pr) 2 Pr)-CHCHCH(n-pentyl). or –CH2CH2CH(n-pentyl)2.
2 a C alkenyl 7. 7. The compound The compoundof of anyany oneone of of claims claims 1 to 1 to 6, 6, wherein wherein Z² Z is is a C19 alkenyl containing one or containing one or two two double doublebonds. bonds.
2 8. The compound of claim 7, wherein Z is 8. The compound of claim 7, wherein Z² is -– (CH2)9CH=CHCH2CH=CH(CH2)4CH3. 2019351809
(CH)9CH=CHCHCH=CH(CH)4CH].
9. 9. A compound A compound selected selected from: from:
0
112 -
Claims (6)
- 2019351809 01 Apr 2025NN N N N O 2019351809ON N O ON N O O ON N N N O Oor a salt or a salt thereof. thereof.10. 10. The The compound compound of anyof anyofone one of claims claims 1 to 1 to 9, 9, wherein wherein the compound the compound is is in the in the form form ofofa apharmaceutically pharmaceutically acceptable acceptable salt. salt.11. 11. The The compound compound of anyof anyofone one of claims claims 1 towherein 1 to 10, 10, wherein the compound the compound is is in the in the form form ofofa acationic cationiclipid. lipid.12. 12. A lipid A lipid particle particle comprising comprising a neutral a neutral lipid,aalipid lipid, lipid capable of reducing capable of reducingaggregation, and aggregation, and a cationic a cationic lipid lipid of claim of claim 11. 11.13. 13. The The lipid lipid particle particle of of claim claim 12,wherein 12, wherein thethe neutrallipid neutral lipidisis selected selected from DSPC, from DSPC,DPPC, POPC, DPPC, POPC, DOPE, DOPE, or the or SM; SM;lipid the lipid capable capable of reducing of reducing aggregation aggregation is a lipid; is a PEG PEG lipid; and andthe lipid particle further comprises a sterol. the lipid particle further comprises a sterol.14. The lipid 14. The lipid particle particle of claim of claim 12 wherein 12 or 13, or 13, wherein thelipid the cationic cationic lipid is is present in present a in a molepercentage mole percentageofofabout about20% 20%andand about about 60%; 60%; the the neutral neutral lipid lipid isispresent presentininaa mole mole- 113 - percentage of of about about 5% 5%totoabout about25%; 25%; thesterol sterolisis present present in in aa mole mole percentage of about about 25% 25% 01 Apr 2025 2019351809 01 Apr 2025 percentage the percentage of to about to about 55%; andthe 55%; and thePEG PEG lipidisis PEG-DMA, lipid PEG-DMA, PEG-DMG, PEG-DMG, or a combination or a combination thereof, thereof, and is and is present in present in aa mole mole percentage of about percentage of 0.5%totoabout about 0.5% about15%. 15%.15. 15. The The lipid lipid particle particle of of claim claim 14,wherein 14, wherein thethe lipidcapable lipid capableofofreducing reducing aggregation aggregationisis PEG-DMG. PEG-DMG. 201935180916. 16. The The lipid lipid particle particle of of claim claim 15,wherein 15, wherein thethe lipidparticle lipid particle comprises comprisesabout about5050 mole%%ofofthe mole thecationic cationic lipid, lipid, about about 10% DSPC, 10% DSPC, about about 38.5% 38.5% cholesterol, cholesterol, andand about about 1.5% 1.5%PEG-DMG PEG-DMG (based (based on 100% on 100% of theof the lipid lipid components components in the in the lipid lipid particle). particle).17. 17. The The lipid lipid particle particle of of claim claim 15,wherein 15, wherein thethe lipidparticle lipid particle comprises comprisesabout about5858 mole%%ofofthe mole thecationic cationic lipid, lipid, about about 10% DSPC, 10% DSPC, about about 30%30% cholesterol, cholesterol, andand about about 2% PEG- 2% PEG-DMG DMG (based (based on on 100% 100% of the of the lipid lipid components components in lipid in the the lipid particle) particle)18. 18. The The lipid lipid particle particle of of any any one one of of claims claims 12 12 to to 17,further 17, furthercomprising comprisingananactive active agent. agent.19. 19. The The lipid lipid particle particle of of claim claim 18,wherein 18, wherein thethe activeagent active agentisisaanucleic nucleicacid acid selected selected from a plasmid, from a an immunostimulatory plasmid, an immunostimulatory oligonucleotide, oligonucleotide, an an siRNA, siRNA, an antisense an antisenseoligonucleotide, aa microRNA, oligonucleotide, microRNA, anan antagomir, antagomir, an an aptamer, aptamer, andand a ribozyme. a ribozyme.20. A pharmaceutical 20. A pharmaceutical composition composition comprising comprising a lipida particle lipid particle of claim of claim 18 or18 19or 19 and aa pharmaceutically and acceptablecarrier. pharmaceutically acceptable carrier.21. A method 21. A method of modulating of modulating the expression the expression of a target of a target gene gene in a in a cell, cell, the the method methodcomprising providing comprising providing to cell to the the cell a lipid a lipid particle particle of claim of claim 18 or 18 19. or 19.22. The The 22. method method of claim of claim 21, wherein 21, wherein the active the active agentagent is a is a nucleic nucleic acidacid siRNA. siRNA.23. A method 23. A method of treating of treating a disease a disease or disorder or disorder characterized characterized by the by the overexpression overexpressionof a polypeptide of a polypeptide in in a subject, a subject, thethe method method comprising comprising providing providing to thea subject a to the subjectpharmaceuticalcomposition pharmaceutical compositionofof claim20,20,wherein claim wherein thethe activeagent active agentisisaanucleic nucleic acid acid selected selected from the group from the groupconsisting consisting of of an an siRNA, siRNA,a amicroRNA, microRNA,and and an antisense an antisense oligonucleotide, oligonucleotide, and andwhereinthe wherein the siRNA, siRNA,microRNA, microRNA, or antisense or antisense oligonucleotide oligonucleotide includes includes a polynucleotide a polynucleotide thatthatspecifically bindstotoa apolynucleotide specifically binds polynucleotide that that encodes encodes the polypeptide, the polypeptide, or a complement or a complement thereof. thereof.- 11424. A method A method of treating a disease or disorder characterized by underexpression of 01 Apr 2025 2019351809 01 Apr 202524. of treating a disease or disorder characterized by underexpression ofa polypeptide a in aa subject, polypeptide in subject,the themethod method comprising providingtoto the comprising providing the subject subject aa pharmaceutical pharmaceuticalcomposition ofclaim composition of claim20, 20,wherein whereinthe theactive activeagent agentis is aa plasmid that encodes plasmid that the polypeptide encodes the polypeptideor a functional or a functionalvariant variantororfragment fragment thereof. thereof.25. A method 25. A method of inducing of inducing an immune an immune response response in a subject, in a subject, the method the methodcomprising providingtotothe comprising providing the subject subject aa pharmaceutical compositionofofclaim pharmaceutical composition claim20, 20,wherein wherein the the 2019351809active agent active agent is isan animmunostimulatory oligonucleotide. immunostimulatory oligonucleotide.26. Use Use 26. of a of a lipid lipid particleasasdefined particle definedininclaim claim1818oror1919ininthe themanufacture manufactureofofa a medicamentforfortreating medicament treatingaa disease disease or or disorder disorder characterized characterized by by the the overexpression of aa overexpression ofpolypeptide, wherein the active agent is a nucleic acid selected from the group consisting of polypeptide, wherein the active agent is a nucleic acid selected from the group consisting ofan siRNA, an siRNA, a amicroRNA, microRNA,and and an antisense an antisense oligonucleotide, oligonucleotide, and and wherein wherein the the siRNA, siRNA,microRNA, microRNA, or or antisense antisense oligonucleotide oligonucleotide includes includes a polynucleotide a polynucleotide thatspecifically that specificallybinds bindstoto aa polynucleotidethat polynucleotide that encodes the polypeptide, encodes the polypeptide, or or aa complement thereof. complement thereof.27. Use Use 27. of aof a lipid lipid particleasasdefined particle definedininclaim claim1818oror1919ininthe themanufacture manufactureofofa a medicamentforfortreating medicament treatingaa disease disease or or disorder disorder characterized characterized by by underexpression ofaa underexpression ofpolypeptide, wherein the active agent is a plasmid that encodes the polypeptide or a polypeptide, wherein the active agent is a plasmid that encodes the polypeptide or afunctional variant or fragment thereof. functional variant or fragment thereof.28. Use Use 28. of a of a lipid lipid particleasasdefined particle definedininclaim claim1818oror1919ininthe themanufacture manufactureofofa a medicamentforforinducing medicament inducingananimmune immune response, response, wherein wherein the active the active agent agent is is an an immunostimulatory oligonucleotide. immunostimulatory oligonucleotide.115 -FIGURE 11.
- 2 Protein FVII Relative 1 1 0.8 Level 0.6
- 0.4 0.
- 4 NO # Day 3 0.20 0.01 0.03 0.01 0.03 0.01 0,03 0.03 0.01 0.03 0.01 0.03PB
- S AF-011-1661 AF-060-1661 AF-060-1661 AF-062-1661 AF-064-1661 AF-064-1661 AF-065-1661 AF-065-16611/8FIGURE 21.4 2) 1.2 Day level 0.8 Protein 0.
- 6 FVII 0.4 Relative 0.2 N1.210.80.60.40.20 0.01 0.01 0.03 0.01 0.01 0.03 0.01 0.03 0.01 0.01 0.01 0.03 0.01 0.03 0.03 0.03 0.01 0.03 0.03 0.01 0.03 0.03 0.03 0.01 0.01 0.03PBSAF-011 AF-054 AF-068 AF-069 AF-070 AF-071 AF-072 AF-073 AF-074 AF-075 AF-076 AF-077;AF-078 PBSAF-011AF-054AF-068AF-069AF-070AF-071AF-072AF-073/AF-074)AF-075AF-076AF-077AF-0782/8FIGURE 3Relative FVII Protein Level ( PBS =1) (PBS=1) = 1.21 10.80.6 0.60.4 0.4 I 0.20 PBS 0.01mg/kg 0.03mg/kg 0.01mg/kg 0.03mg/kg 0.01mg/kg 0.03mg/kg 0.01mg/kg 003mainAF-011-1661 0.03mg/kg AF-011-1661 AF-074-1661 AF-074-1661 AF-082-1661 AF AF-082-1661 AF AF-083-1661 AF AF-083-1661 AF3/8PCT/US2019/053617FIGURE 4Relative FVII Protein Level - * 48 hours 1.614 1.2 22 3 -0.80.60.40.20.005 0.01 0.03 0.005 0.01 0.03 0.005 0.01 0.03 0.005 0.01 0.005 0.01 0.03 0.005 0.01 0.03 0.03 mg/k mg/kg 39PSS PBS AF-070-1661 AF-068-1661 AF-074-1661 AF-073-1661 AF-072-1661 AF-072-16614/8WO 2020/072324 2020/07234 OM PCT/US2019/053617FIGURE S HOGRE51.2 12 All Groups - $ - RES-17-046-C n reLNP NHP Study RES-17-046-Cand1 Pre-Dose) to (Rel. 0.8 Level Plasma F12 90AF-011-167990 (2M/2F) 0.6 AF-070-167990 (3M) (WE) 90 (3c) 066291-620-3V AF-079-167990 (3F) AF-073-167990 (3M) (WC) 0.4 V0 ........ AF-074-167990 (3F) (HC)0.2 200 01- 0 5 9 10 St 15 00 20 25 9Z 30 08 98 35 Study Day5/8 8/£FIGURE 6A ......... G1: AF-011 0.3 mg/kg 1.4 1.4 G2: AF-094 0.03 mg/kg G3: AF-094 0.1 mg/kg 1.2 G4: AF-094 0.3 mg/kg D0) Pre-Dose to (Norm Level Plasma F12 Rel. 1.00.03 mg/kg 0.80.6 0.1 mg/kg 0.40.2 0.3 mg/kg0.0 -5 5 15 25 Study DayFIGURE 6B********* G1: AF-011 0.3 mg/kg 1.4 G5: AF-079 0.3 mg/kg1.2 1.21.0 D0) Pre-Dose to (Norm Level Plasma F12 Rel. 0.80.60.4T 0.2 0.3 mg/kg0.0 -5 5 15 25 Study Day8/9 6/8PCT/US2019/053617FIGURE 7G1: G1: AF-011 AF-011 0.3 0.3 mg/kg mg/kg 1.4 G6: G6: AF-073 AF-073 0.03 0.03 mg/kg mg/kg G7: G7: AF-073 AF-073 0.1 0.1 mg/kg mg/kg 1.2 G8: G8: AF-073 AF-073 0.3 0.3 mg/kg mg/kg D0) Pre-Dose to (Norm Level Plasma F12 Rel. 1.0 t a 0.8 0.03 mg/kg I 0.60.4 0.1 mg/kg0.2 0.3 mg/kg0.0 0.0 -5 0 5 10 15 20 25 Study Day7/8FIGURE 8G1: AF-011 0.3 mg/kg 1.4 G9: AF-093 0.03 mg/kg G10: AF-093 0.1 mg/kg 1.2 D0) Pre-Dose to (Norm G11: AF-093 0.3 mg/kg Level Plasma F12 Rel. 0. 00.80.60.03 mg/kg 0.40.2 0.1 mg/kg0.3 mg/kg 0.0 -5 5 15 25 Study Day6 PIGURE FIGURE 9G1: AF-011 0.3 mg/kg 1.4 G12: AF-083 0.1 mg/kg 1.2 G13: AF-083 0.3 mg/kg D0) Pre-Dose to (Norm Level Plasma F12 Rel. 1.01 1.0T OT0.80.60.40.2 0.1 mg/kg PH 0.3 mg/kg 0.0 0.0 -5 -5 5 15 25 Study Day8/8
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| ES2951836T3 (en) * | 2018-10-01 | 2023-10-25 | Alnylam Pharmaceuticals Inc | Biodegradable lipids for the delivery of active agents |
| CA3137956A1 (en) * | 2019-04-25 | 2020-10-29 | Intellia Therapeutics, Inc. | Ionizable amine lipids and lipid nanoparticles |
| EP4255888A4 (en) * | 2020-12-02 | 2024-12-04 | Merck Sharp & Dohme LLC | LIPID NANOPARTICLE COMPOSITIONS CONTAINING CATIONIC MONOESTER LIPIDS |
| WO2022155728A1 (en) * | 2021-01-19 | 2022-07-28 | The University Of British Columbia | Sulfur-containing lipids |
| CN115010681A (en) * | 2021-09-07 | 2022-09-06 | 安徽荣灿生物科技有限公司 | Cationic liposome molecule and preparation and application thereof |
| CN115894281B (en) * | 2021-09-22 | 2025-07-01 | 广州谷森制药有限公司 | Cationic lipid compound, preparation method, composition and application thereof |
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